jan_2007

Editorial Board :

Chief Editor :Dr. K.V. Somasundaram

Editor :Dr. J.S.N. Murthy

Joint Editor :Dr. J. Damodaran

Members :Dr. Padma Srikanth

Dr. S. Shobana

Dr. Emmanuel Bhaskar

Dr. B. Harapriya

Dr. Amarender Vadivelu

Mr. N. Venkatesh

Mr. A. Selvam

Mr. R.C. Perumal

Dr. Solomon F.D. Paul

Secretarial Assistant:Ms. M. Viji

SRI RAMACHANDRA JOURNAL OF MEDICINEVOLUME - 1, ISSUE - 2, JANUARY 2007

CONTENT

Published by :THE REGISTRAR

SRI RAMACHANDRA UNIVERSITYPorur, Chennai - 600 116, INDIA.

Statements and information contained in this publication are believed to be reliable. The opinions stated in these articles areexpressions of the authors and not necessarily those of the editors and members of the editorial board. All rights reserved.No part of the publication can be reproduced without prior permission of the publisher.

From the Editors Desk - iii

Our Scientific Reviewers - iv

“Rasipurathu Nayagan” - Dr. D. Gnanaprakasam - v

On Higher Education - Dr. K.V. Somasundaram - vi

Photos Gallery - viii

Invited ArticleCreating Prodev – A Program to Formalize the Informal Curriculum - 1

Special ArticleInterventional Radiology – Speciality of the Current Century:Initial Experience at SRMC & RI (Part II continued...) - 4

Original ArticleClinico – Mycological Profile of Dermatophytic Skin Infectionsin a Tertiary Care Center – A Cross Sectional Study - 12

Labelling Transformed Mitotic Cells with Fluorescent Antibody Conjugate - 16

Brief ReportChromosome Painting and its Versatility in Modern Diagnostics - 20

Review ArticleLaparoscopic Bile Duct Injuries – Controversies and Consensus - 27

Pharmacogenomics – A New Perspective in Clinical Healthcare - 34

Short ReviewPsychological Autopsy: the Psychological Assessment of an equivocal Death - 41

Pulse Therapy in Dermatology - 44

Case ReportTooth Within a Tooth - 47

Cortical Venous Thrombosis in a Case of Preeclampsia - 50

Orbital Complication of Paediatric Sinusitis - A Case Report - 52

Laparoscopic Nephrectomy in Children:A Case Report and Review of our Experience - 54

Acrodermatitis Enteropathica – A Case Report - 57

Unusual Presentation of Rhinosporidiosis - A Case Report - 60

A Case of Reactive Thrombocytosis - 62

Address for correspondence: Office of the Editor, Sri Ramachandra Journal of Medicine,Sri Ramachandra University, Porur, Chennai - 600 116. www.srmc.edu/srjm

Scope of the Journal

The Sri Ramachandra Journal of Medicine - a scientificjournal, entertains communications on all aspects of originalbiomedical research contributing to the advancement ofknowledge in medical sciences. The scope of the journalallows publication of papers on medical educationat undergraduate and postgraduate levels in eithermedical or paramedical courses; innovations in techniques;epidemiologic investigations and case reports. Readers areencouraged to write comments on papers published in thejournal in the form of correspondence. Brief communicationcontaining significant findings will be given priority.Review articles are also invited on topics of currentinterest. The journal is issued thrice in every calender year.All papers are subjected to peer review by the EditorialBoard and also experts in the field before acceptance forpublication. All papers are accepted subjected to editorialchanges.

Articles submitted to the journal should abide by thefollowing manuscript submission guidelines.

Submission of Manuscript :

Each manuscript submission should include thefollowing documents.

Part I - Title PagePart II - Manuscript filePart III - Acknowledgment, declaration by authors,

patient consent and supplemental file.

All contents related to manuscript submission shouldbe in English on a White paper of A4 size ( 210 x 297) withmargins of 25mm (1 inch) wide on all the four sides. Printshould be on oneside only with double spacing throughout.Pages should be numbered consecutively, beginning withtitle page. Lettering should in Times New Roman with afont size of 12. Three copies should be submitted to theeditor. A copy of the title page and manuscript file must beemailed (as an attachment) with a covering letter address tothe editor.

PART I - Title Page must include : a) Title of the article b)Name of each contributor with the highest degree andinstitutional affiliation. c) Name, cellphone, e-mail of thecorresponding author.

PART II - Manuscript file : Should include the text of thearticle followed by tables and figures. The table/figure number(eg: Table 1, Figure 1) should be appropriately mentioned inthe text. The references should be numbered as they appearin the article and must be written in Vancouver style. Thereferences should be kept after the tables/figures.

PART III - Acknowledgment: May include the names withdetails of affiliation, if any. They will appear in the article,but before the references.

Declaration by the authors: All the authors should submit adeclaration regarding originality of the work, submission toother journals, whether the articles were already publishedand financial conflicts of interest which might influence themanuscript.

Supplemental file: These articles / texts which might help thereview process, they should be relevant to the article submitted.

Nature of Articles - 1. Original articles: Articles of originalresearch are welcome in this category. Articles should notexceed 4000 words. It must include an abstract of 250words which should be structured as a) Aim of the study,b) Methodology, c) Results and d) Discussion. Minimumof three MesH words to be mentioned at the bottom of theabstract. Upto 50 references may be included in thesearticles.

2. Review articles: These articles addressing an issue /theme of current interest. They should not exceed 4000 words.Should include an unstructured abstract of 400 words withthree MesH words. Article may include upto 100 references.

3. Case reports: Case reports reflecting a major clinicalproblem are welcome for this section. Word count shouldbe restricted to 300 with references upto 5. May include 2photographs and 1 table. Photographs having visibleidentification of patients must have written consent fromthe patient/close relatives. Case reports having more than 1case will be given preference. Photographs should be at least5 by 7 inches. Photographs may be submitted in a digitalfile, preferable in a JPEG (or) Tiff format. Photographs shouldbe labeled appropriately.

4. Letter to the Editor: Correspondence to the editorregarding an article published in the journal are invited inthis category. The content should be restricted to 300 wordswith references upto five.

GUIDELINES FOR AUTHORS

From the Editor’s Desk

It was a moment of joy for the editorial board

when the maiden edition of Sri Ramachandra Journal

of Medicine was released in September 2006.

We had apprehension and fear about the outcome

and response from the readers. Fortunately, it was

positive and encouraging from every quarter of our

university and other major educational institutions.

It was delightful experience to get constructive

comments, words of encouragement for the first

issue.

With the good wishes and encouragement of

the management and senior faculty member, we are

venturing with the next issue. This issue includes

the Sri Ramachandra experience to formalize the

informal curriculum using an innovative method to

impart “professional values”, the concluding part of

the article on interventional radiology, the application

of use of fluorescent antibody conjugate in labelling

transformed mitotic cells with the possibility of

exploitation of these antibodies for cancer detection,

review articles and case reports. We are aware of

our responsibility and we in the editorial board hope

to live up to your expectations.

This second edition is in front of you

with untiring work done by all my colleagues

under the watchful eyes of the Chief Editor

Dr. K.V. Somasundaram. I will be failing in my duty

if I don’t highlight the silent, efficient, hard work

put up by Mrs. Viji of the Dean’s office. I hope this

edition will draw the attention of many more of our

readers and give us their suggestions and constructive

criticism to enable us to improve further.

J.S.N.MURTHYEditor,

Sri Ramachandra Journal of MedicineSri Ramachandra Medical College & Research Institute

Sri Ramachandra UniversityPorur, Chennai - 600 116

email : [email protected]

Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007 iii

Dr. Betty Chacko

Dr. K. Balaji Singh

Dr. Georgi Abraham

Ms.Gita Menon

Dr. T.R. Gopalan

Dr. Hannah.R. Vasanthi

Dr. M. Jayarajah

Dr. S.B. Jothi Ramalingam

Dr.Jyothi Somani

Dr. Kalpana Balakrishnan

Dr. Kalpana Suresh

Dr. T.S.Lokeswari

Dr. Mahesh Vakamudi

Dr. Mahalakshmi Veeraraghavan

Dr. M. Mallika

Dr. S. Manjunath

Dr. Norbert L. Wagner

Dr. L.N. Padmasani

Dr. N. Palaniappan

Dr. D. Prathiba

Dr. Preetam Arthur

Dr. Radha Kumar

Dr. S. Rajendiran

Dr. Ramya

Dr. A. Ravikumar

Dr. A. Rekha

Dr. Sandhya Sundaram

Dr. Sanjeeva Reddy

Dr. Sanjeev Mohanty

Dr. B.W.C.Sathiyasekaran

Dr. S. Seethalakshmi

Dr. Shanthi Vijayaraghavan

Dr. M.Subramaniam

Dr. M.K. Sudhakar

Dr. R. Suresh

Dr. Uma Sekar

Dr. Vasantha Janardhan

Dr. D. Vasudevan

Dr. V. Vedagiri

Dr. P. Venkatachalam

Mr. N. Venkateswaran

Dr. Vinod Kumar Panicker

Dr. Viswanath.M.Pai

OUR SCIENTIFIC REVIEWERS.....

The Editorial Board gratefully acknowledges their contribution.

iv Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007

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Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007 v

ON HIGHER EDUCATION …..

Dr. K.V. SomasundaramDean of Faculties

Education is an inclusive process; is enrichinglife; formally or informally, every experience is aneducation. Every event in life, every moment ofliving is an education.

Accordingly, medical education, at the firstinstance, should create an individual who will be amedical person. Corollary, that person shouldcontinue to be educated as anyone else, as anindividual and as a professional. The period spentin the university as Under-Graduate or Post-Graduateshould help to evolve this total, complete person.

India’s first national policy on Education wasannounced in 1968. After periodic minor changes,the next one was announced 18 years later.Concepts on education are evolving at a very highspeed and our policies should accommodate them,adapt and develop with a much greater speed andpower. For historic reasons, English has spread inour country – this is a blessing, though a legacy ofcolonial rub. And this is an important base forcreating a knowledge power house.

Tracing the history of medical education inIndia, one becomes aware of what was practisedfew thousands of years before. Very many of themare in vogue today with English equivalents likepersonality of the practitioner, character, innovation,learning by roll- model, women’s role etc., As youread and understand these works, you are spurredto greater heights.

Today knowledge proliferates but wisdomlanguishes. The real education is the education byvalues.

With enough background material andguidelines as advocated by UNESCO, a system hasalready been conceptualized. This consists of threelayers.

The formation layer aims to build the personalitybased on values and enables continuous learning.This includes values which are social, economics,wellness, citizenship, humanities and aesthetics,moral and spiritual.

The next layer should help one “Learning tolearn” and “Learning to do”. This consists mostly ofskills – like learning from different sources, sensoryinputs, studying, communication includinglanguages, expression in different situations etc;technology improvement, personality and skills forliving life as it should be.

The third layer should facilitate transformationinto “Learning to live”. This has a myriad topics likework culture, Total Quality Management,competitiveness, global approaches,entrepreneurship, corporatisation, professionalism,innovation or thinking differently, attitudinal changesand certain autonomy.

To achieve this aim, the faculty should betrained. In the first year, teachers should trained inteaching- learning process and their skills should becredentialled. The present national process fortraining teachers may need modification beforeapplying to health professionals. Over a period,mentoring is necessary for the teachers also.Research is a big topic in India today. It is anexciting field. There are lots of money for the futurewhich could make this country one of the frontlinersin medical innovations like biotechnology ornanotechnology. The assessment of the teachershould be on well laid principles and the concernedindividual should be aware of it. Assessment fromdifferent sources should be encouraged. Today theyalso need to be trained in basic administration. Asystem of education should have a good assessmentprocedure in place. We need to be more practical.Teachers, particularly assessors should be motivatedand supported well financially. Motivation shouldbe altruistic and not for pride or prestige.

Out students are going to be professionals. Sothe curriculum should include professionaldevelopment as important segment, atleast in theperiod of resident training. This should encompassaccepted principles like development as anindividual, personality, character, skills in

vi Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007

management of time, people, finance, stress andstrain of living, active listening, body language, etc.,One should not forget gender issues, customerexpectations, stake holder’s demands, civil law, ethicsand good laboratory practice.

One is reminded again that it is in thisprofession, globally, that we have maximum numberof suicides, alcoholic and depressions. Why is it?Have we failed to train them properly? Obviouslyemotional development has not been stressed asmuch as being intelligent. Gandhiji said “Informationwithout formation leads to deformation”. One shouldalso remember what former Prime MinisterMr.Jawaharlal Nehru famously described as“Scientific temperament”.

The student should have interest developed in“learning” as an art, to be practised continuously,curiosity of the childhood should become an“enquiring mind” and “discerning eyes” of the future.One should also practise to unlearn what is obsoleteand relearn regularly. For the professional topractice, can we not apply a compulsory periodicassessment to make them really worthy of the trustbased on them by the society?

Our training of residents leaves much to bedesired. Currently they learn to practice what theyhave learnt, under supervisors. This is good and entailsthem for registration. But it is just not enough. Theyshould have diversity or a choice depending on whatthey want to be next step. And there should be anassessment at the completion of the period, reflectedin a comprehensive certificate.

What with WTO and GATS, there is a track intranslational higher education, currently a hot issue.Is education still a service, a calling or just another

profession? If the students and society becomecustomers, then it becomes a trade.

We should be able to think differently, not beafraid of a change. That is not fundamentalism orextremism. As aptly put by many, we are oftenprisoners of our own thoughts. The inputs are frommany and variable sources, often not tested orchallenged. Thought is just a network, made ofthreads. Not being afraid of the unknown, we shouldlearn to think differently and constructively bothas an individual and as a group. This is just asadvocated by many as a useful tool, a must for afuture – future is not some distance away, it is whatwe create today.

The curriculum with due checks and assessmentprocess should change to include contemporaryissues like related civil law, consumerism, ethicsetc.,

There are quite a few lacunae today.Importantly there is very little study or research toassess the utility of what is in the curriculum in theimmediate period – internship; during P.G.programor while doing professional work. Does a very highacademic record means he will be a highlycompetent professional, a good individual. It isnecessary to be a professional. It is a verycomprehensive term. It is essential today especiallysince expectations and awareness are increasing; itis highly error-prone service. Health care is veryexpensive. It is a truly global scenario.

Research is an integral part of higher education,big or small, then should be one project atleast andscientific writing and speaking should be developed.

A sentence needs a full stop to be meaningful;but education has no full stops.

Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007 vii

Prof. S. Rangaswami appointed Vice-Chancellor, being felicitated by the Chancellor

Prof. S. Rangaswami’s Inaugural address as Vice-Chancellor

Photo Gallery

viii Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007

Life Cell and Sri Ramachandra “TRICELL” Inauguration by Chancellor

vi

Photo Gallery

Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007 ix

“Horizons” - Dr. Amit Patel, Director, Cardiac Stem Cell TherapyPittsburg University

Photo Gallery

Active Participants

Harvard Medical International - ‘Team for Patient Safety’

Globalisation of the Team

HMI - Sept. 2006 @ Sri Ramachandra

x Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007

Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007 1

CREATING PRODEV – A PROGRAM TO FORMALIZE THE INFORMAL CURRICULUMKrishna G Seshadri

BACKGROUNDOne of the most intriguing aspects of becoming a

physician is learning the skills that many educationists andexperts club together in a rather unwieldy basket calledthe “hidden curriculum”. The word intriguing is useddeliberately in this context because many aspects of thenature and the acquisition of the hidden curriculum areindeed shrouded in intrigue. Anyone who examines themedical profession from a good objective distance is sureto be amazed that this profession spends an enormousamount of time in instructing its apprentices on the morecapricious aspects of its trade, for instance the choice oftherapy for Chronic Myeloid Leukemia which has changedat least twice in the last ten years1 and little or no time atall in the skills that will probably remain unchanged forthe rest of the learner’s career for example communicationwith a patient. From a faculty perspective even moredangerous is the knowledge that “the hidden curriculumof rules, regulations, and routines is transmitted mostly byresidents (rather than the faculty) in clinic hallways andthe hospital, often late at night when residents and studentsare on call”2,3

For a major part of the last century, the acquisition ofthe hidden curriculum – which for the purpose of this articlewe will call professionalism (although this does include anumber of skills that does not strictly fall into this category)was relegated to an ill defined osmotic process called“role modeling”. Unfortunately as Stern elegantly pointsout, role modeling is in the eye of the beholder – the studentand not the teacher. “Individuals who are seen as mentorsmay not realize that they are teaching professional valuesand those not seen as mentors may believe they are.”

Since the 1970s many medical schools around the worldhave developed modules or content areas that teach as theleast, medical ethics. National committees that designcurricula have acknowledged the need to formalize the“informal curriculum.” Several excellent examples ofattempts by prominent medical schools, to create anenvironment of acquisition of, and the reflection on these“professional values” by medical students have beenpublished. These innovative methods have in part beenspurred on and sustained by a recognition by regulatory andlicensing agencies in various nations (including the Medical

CORRESPONDING AUTHOR :

Dr. KRISHNA G SESHADRIProfessor and Head,Department of Endocrinology Diabetes and MetabolismSri Ramachandra Medical College and Research InstitutePorur, Chennai - 600 116.email : [email protected]

Council of India4) that these skills must be an importantpart of the core curriculum.

THE SRI RAMACHANDRA EXPERIENCE

The curricular innovation initiative can be traced backto the conduct of the “Status of Progress of Reforms inUndergraduate Curriculum and Education (SPRUCE)” aconference conducted by our university in the year 2000with the support of the Medical Council of India. Theconference highlighted that there was a common aspirationamong the many stake holders of education in India increating a more progressive patient centered problem basedcurriculum with clear emphasis on professionaldevelopment. Following this Sri Ramachandra University(SRU) created a curriculum development initiative with aview to create a competency based curriculum by the year2008.

The curriculum development group developed aunique process of working backwards –first identifying theknowledge, skills and attitudes that a physician needs topossess while he or she practices in a particular domainafter graduation and then place them accordingly into thevarious phases and departments. The cross content areaswere marked for vertical or horizontal integration. It wasapparent during this process, that a large number ofcompetencies that would in the past be left out in a greyzone to be acquired informally will now have to beformalized into the curriculum. As the curriculumdevelopment process proceeded, the group discovered thatthis basket was rapidly topping up and needed to be addressedin a fashion that will be complementary to the newcurriculum. Further direction to the curriculum developmentprocess was given by the leadership of the Sri RamachandraUniversity which expressed a desire to see “students fromSri Ramachandra graduate with real life skills”.

The curriculum group felt that this was an opportunityto develop a unique model to impart professional skills –and PRODEV was born.

DEVELOPING PRODEV

The first step in creating PRODEV was to establish aneeds document (table 1). A faculty questionnaire wascirculated that asked the respondents to indicate if they feltif formal instruction was desirable in seventeen areas of theinformal curriculum. Except for “alternate medicine” forwhich there was minor disagreement, the overwhelmingresponse for each of these questions was one of stronglyagree or agree. Several focus groups were also held withvarious levels of students to ascertain their views.

Invited Article

2 Sri Ramachandra Journal of Medicine Vol. 1 Issue 2 January 2007

Once the need was established, content areas wereidentified (table 2) and a debate initiated on who shouldteach these content areas. There are several reports on contentareas in the literature, the most prominent of them beingthe Pond report5 which suggested the identification of agroup of experts to teach medical ethics. It was clear fromthe questionnaire submitted to the faculty that a majorityof faculty professed no experience or comfort in teachingprofessionalism formally. However it was evident that faculty

Invited Article

Table 1. Steps in Developing PRODEV

Step Methodology1. Establish Faculty questionnaire,

need Student Focus groups

2. Content Literature review, Discussion withidentification peers from other institutions,

Validation by faculty questionnaire

3. Structure Consensus by curriculum groupdevelopment to create a hybrid program (see text)

4. Faculty Formal day long faculty developmentdevelopment program

5. Content Case writing, identification of keydevelopment learning articles, identification of

important learning sites on theworld wide web

6. Student Formative bimonthly assessment,assessment MCQs Creative writing and Ethics

and communications OSCE

7. System Formal bimonthly feed back fromassessment faculty and students. Half yearly openfor CQI house with faculty and students

Table 2. PRODEV content areas1. What is expected of a doctor in the 21st century2. Foundations of Medicine and the law3. Foundations of Medical Ethics4. Reasoning in Clinical Practice5. Introduction to Clinical Research6. Ethical Aspects of Human Research7. Taking the bench to the bedside8. Effective Communication9. Connecting with Patients10. Working in a Health Care Team11. Etiquette for Doctors12. Health Care Systems13. Quality in Health Care14. The Economics of Health Care15. Alternate Health Care Systems16. Personal Development for Professional Growth17. Preparing for Lifelong Learning

cutting across departmental and hierarchal lines were eagerand willing to be part of the process (except one, all therespondents expressed willingness to be part of this process).It was fortuitous that the program chose interested novicesthan confine this to the hands of a small group of skilledexperts as will be evident below.

From the outset, it was clear that the process toimplement PRODEV needs to be longitudinal, spread acrossthe five and a half years, must straddle departmental silosand must be taught by any faculty who are willing to teach.Departments willingly donated surplus time for the program.Three hours (one in the morning and two in the afternoon)once in two weeks were made available for PRODEV). Oncethe time was allocated it was decided to start the programwith the students in the 8th semester progressively movingto lower phases each year depending on the success of theprogram.

CREATING A STRUCTURE FOR PRODEV (Fig. 1)A hybrid method of instruction was evolved – the

discussion would revolve around a case introduced to a smallgroup by a facilitator; the group would identify learningobjectives and do self study and reflect for two weeks. Ananchoring lecture would then be delivered by “an expert”on the morning of the discussion. The patient problem wouldbe discussed by the small group in the afternoon and thecase resolved.

Figure 1. Structure of Prodev

After the completion of several blocks of cases – anassessment would be conducted both formative basedon a standard form developed by the curriculum groupas part of the curriculum development initiative as wellas a summative test that consisted of 1) a morning writtensession consisting of MCQ s and a creative writing essayand 2) an afternoon session with an ethics or acommunication OSCE.

IMPLEMENTING PRODEVIn January 2005 the first faculty development for

PRODEV was held for sixty faculty members. Three facultymembers were assigned to each group to be facilitators.The expectation was that the faculty will not only help thesegroups with PRODEV but also evolve into mentors for the


students. The program was started with the incoming8th semester students and extended to the sixth semesterstudents in July 2005. The first PRODEV case wasprovocatively called “How to buy a kidney”. Two batchesof medical students have completed PRODEV.

OUTCOMESPRODEV has been a gratifying experience for its

creators, facilitators and students. An analysis of studentand faculty feedback reveals that PRODEV has realizedmost of its intended outcomes; in addition realizedseveral unintended but desirable outcomes. These aresummarized in table3.

Table 3. Benefits from PRODEV – based on responsesobtained in written feedbackFor the Students1. “Learnt how to talk to patients”

2. “Learnt to face real life situations”

3. “Were able to interact with faculty in an informalatmosphere”

4. “Were able to learn from our colleagues”

For the faculty1. “Widened our horizons”

2. “Learnt about topics we were not too comfortable with”

3. “Were able to work with colleagues from otherdepartments”

For the Curriculum process1. A preview of the integrative curriculum

2. A situation were faculty were able work across silos

3. A test for seeing how students use reflection andintrospection

4. A test of collaborative and group learning

For the curriculum group, PRODEV was an interestingtest run of the new curriculum – a testing laboratory to findout what will be the consequences of introducing a hybridprogram, how faculty work across departmental silos, howstudents migrate from an environment of emphasis on roteknowledge to an environment where a larger part of thelearning responsibility will rest on them. The resultsemboldened the curriculum development group to roll outthe preclinical integrated curriculum a year ahead ofschedule.

Student and faculty involvement have been the key tothe success of the program and the intensity of theirinvolvement has carried the program forward. It is toopremature to ascertain the long term gains in the career and

professional lives of students but a random survey of thefirst batch of students who are currently in their CRRI yearreveals that they feel better equipped to handle “every daymedicine”.

CHALLENGES THAT REMAINWhile PRODEV has unfettered acceptance in the

institution – it still faces the challenge of not being anintegral “core of the curriculum”. A decision was made tonot extend PRODEV into the 9th semester as students andsome faculty perceived that this would be a distraction in asemester that student attention must be focused on theirfinal examination. Unless the content areas championed byPRODEV are an integral part of this examination the attitudetowards professional development sessions will remain, andbe an issue that will need to be addressed as we gain moreexperience and more support from regulatory agencies likethe Medical Council of India.

CONCLUSIONPRODEV has been a successful attempt at introducing

a longitudinal multidisciplinary professional developmentprogram for MBBS students. Its success has paved the pathfor the implementation of an integrated competency basedpatient centered curriculum in the university.

ACKNOWLEDGEMENTSThe author wishes to acknowledge the contributions of

the leadership, the curriculum development group, thePRODEV coordinators, the medical education unit, facultyand all the students who made PRODEV possible.

REFERENCES:1. Goldman JM, Melo JV. Chronic Myeloid Leukemia– Advances in Biology and New Approaches to Treatment.N Engl J Med. 2003; 349: 1451 - 64

2. Stern DT. Practicing what we preach? An analysis ofthe curriculum of values in medical education. Am J Med1998; 104"569 -75

3. Stern DT. In search of the informal curriculum: whenand where are professional values taught? Acad Med 1998;73 Suppl 10 S28 –S30.

4. Medical Education: The Developing physician –Becoming a professional Ster D.t., Papadakis M. N Engl JMed 2006;355:1794-1799,Oct 26, 2006.

5. Rules and regulations of the Medical Council of India.Accessed January 2007

6. Report of a working party on the teaching of medicalethics. British Institute of Medicine. 1987; London: TheInstitute

Invited Article


Key words : Interventional Radiology

INTRODUCTION:Radiology remained a diagnostic science ever since

its inception following the discovery of X-rays by Roentgenin 1895. The speciality has grown with contrast studies,fluoroscopy and invasive access to blood vessels bySeldinger’s technique which have paved a new way toapproach clinical diagnosis. The term “interventionalradiology” was coined by Alexander Margulis in 1960 todenote a group of procedures done with the help ofimaging. Initially it began with simple image guidedbiopsy or abscess drainage with the help of fluoroscopy.The addition of cross sectional imaging in the last twodecades such as CT, Ultrasound and MRI has provided anew dimension in understanding disease process. Theinnovation of angioplasty (PTA) by Charles Dotter in 1964was a break through in interventional radiology. Slowlybut inevitably procedures that once required surgeons andsurgical incisions have been replaced by percutaneous imageguided techniques. With the addition of technologicalinnovation and micro devices, the scope, and the numberof procedures have increased with a separate specialitybeing started for each organ/system of the body. Theaddition of 3D rotational angiography was a boon to theinterventional neuroradiologist. Basically interventionalprocedures aim at opening or closing, ie., opening of ablocked duct or blood vessel and closing of an abnormalor leaking duct or vessel by introduction of various devices.This article aims at the scope of interventional radiologicalprocedures, including those performed at SRMC & RI.

MATERIALS AND METHODS:Invasive angiography and interventions performed by

the interventional radiologist has been analyzed. A totalof 2732 angiographic / interventional procedures wereperformed (M=1737: F=995; Age range=1day to 87years,N = 48years). The procedures were performed usingBiplane DSA 3D rotational angiography system (LCN+GE Milwaukee). The indications for interventionalprocedures included acute vascular emergencies, controlof acute bleeding, acute stroke, acute limb, several difficult

INTERVENTIONAL RADIOLOGY – SPECIALITY OF THE CURRENT CENTURY:INITIAL EXPERIENCE AT SRMC & RI

Part II continued...Santhosh Joseph a, Deepak Bharathi a, Senthilnathan a


Dr. SANTHOSH JOSEPHProfessor and HeadDepartment of Radiology & Imaging SciencesSri Ramachandra Medical College and Research Institute,Porur, Chennai – 600 116.email : [email protected] Department of Radiology & Imaging Sciences

Special Article

clinical problems such as AVM, aneurysms etc. and forpreventive measures, to manage therapeutic complications,as an aid to palliative care, pain management andinfertility. This article aims to highlight the variety andthe scope of these procedures in patient care.

E) INTERVENTIONS IN CHRONIC PROBLEMSE.1 Hypertension - Intervention for renal artery stenosis

(Fig 15)There are many causes for obstructive lesions of the

renal artery, common ones being atherosclerosis (90%) andfibromuscular dysplasia (FMD). The clinical manifestationsof renal artery occlusive disease are hypertension, renalfailure or both. Hypertension secondary to renal arterystenosis accounts for 1-5% of patients with hypertension.Angioplasty and stent placement are the percutaneoustechniques commonly used in the treatment of obstructivelesion of the renal artery

A B C

Fig. 15 Renal PTA and Stenting.

Renal PTA and Stenting: Patient presented chronichypertension. A: Aortogram reveals significant bilateralproximal renal artery stenosis. B: Restoration of flow inboth the renal arteries following bilateral stent placement.C: Stents in situ.

Chart 1: Diagnostic and Interventional procedures

Chart 2: Number of procedures


Renal artery angioplasty (Fig.15) and stent placementis a challenging procedure and depends on the types andlocations of lesions, the size of the vessels and the anglesbetween the aorta and the renal arteries. Metallic stentsimprove the technical and clinical outcomes inatherosclerotic disease, particularly for ostial lesions. Unlesscomplicated by a dissection, stents are not necessary formost forms of FMD.

E.2 Claudication / Chronic limbThe prevalence of atherosclerotic peripheral arterial

disease increases with age from 3% in individuals aged40-59 to 20% in older than 70 years. Most commonsymptom is pain upon ambulation (claudication). Asmaller percentage have rest pain, tissue loss or gangrene.A wide variety of technologies have been applied to thisvascular bed including angioplasty, stents, stent – grafts,mechanical atherectomy and laser atherectomy. Occlusivedisease of the SFA and popliteal artery can be effectivelytreated with angioplasty when the stenoses or occlusionsare focal (Less than 5cm in length). The technical successrate for percutaneous SFA and popliteal interventions isgreater than 95% for stenoses and 85-90% for occlusions.Similar interventional techniques are also available forupper limb and visceral arteries. The major advantage ofendovascular therapy includes repeatability in primary orsecondary failure whereas redo procedures are cumbersomein surgery.

F) INTERVENTIONS FOR THERAPEUTIC COMPLICATIONS

F.1 Intravascular foreign body removalThe most common cause for accidental embolisation

is transection of an indwelling polyvinyl or polyethylenecatheter when it is withdrawn across a sharp needle bevel.Intravascular foreign bodies can cause serious complicationssuch as death, transient arrhythmia, sepsis, thrombus andpulmonary emboli. These can be successfully removed usingretrieval devices such as the loop snare, hooked catheter,helical basket, hook guidewire etc.

G) INTERVENTION IN DIFFICULT CLINICAL SITUATIONS:G.1 Carotid cavernous fistula (Fig. 16)

These are spontaneous or traumatic connectionsbetween the carotid artery and the cavernous sinusand can be classified as direct or indirect. The traumaticcarotid cavernous fistula occurs following head injury wherethere is a tear connecting the carotid artery and cavernoussinus, whereas spontaneous CCF are dural AVM’s at thelevel of the cavernous sinus. Reversal of direction of theflow through the ophthalmic veins and or spheno-parietalsinus is possible if an arterio-venous connection developsin the cavernous sinus. Elevated venous pressure in theveins draining the orbit may produce orbital venouscongestion, transudation of interstitial fluid, proptosis,increased intra ocular tension and secondary glaucoma. Lessfrequently reversal of flow into the sphenoparietal sinus withresultant cortical venous hypertension poses a risk of intra-cerebral hemorrhage and warrants emergent therapy.

Endovascular treatment options available includetransarterial balloon embolisation for symptomatic directCCF and trans arterial or transvenous coil embolisation incase of indirect CCF.

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A B C

D

E

Fig.16: Carotid Cavernous FistulaCarotid Cavernous Fistula : A 24year old lady presentedwith proptosis, chemosis and ptosis of the right eyefollowing RTA. A: Right carotid angiogram showing carotidcavernous fistula. B: Following embolisation of CCFwith balloon absence of SOV filling. C: Balloon in situ.D: Pre embolisation clinical picture. E: Post embolisationshows normal appearance.

G.2 Dural AVM (Fig.17)Intracranial dural AV Fistulae are acquired arterio

venous shunts located inside the duramater. They accountfor 10-15% of all intracranial arteriovenous lesions.Their presentation and prognosis are variable, symptomsare due to arterialization of the venous system. Endovasculartreatment is particularly tricky. The choice of treatmentdepends on the natural risk of the disease which may beestimated for each patient according to the type of venousdrainage. The possible modes of treatment include arterialembolisation with particles or glue, sinus occlusion withcoils or even direct puncture and coil embolisation. Themajor symptoms in patients with dural AV fistulas includetinnitus, proptosis, raised ICP or IOP and mentationchanges. The transvenous embolisation or direct punctureembolisation through a burr hole provides a new opportunityto treat these highly complex and challenging problems withgood clinical results.


A B C

D

E

Fig. 17: Dural AVMDural AVM: 43 year old gentleman presented withproptosis, chemosis of the right eye with right third nervepalsy. A: Right carotid angiogram showing dural AVM atthe level of the right transverse sinus. B: Coil mass in situin the right transverse sinus.C: Post coil embolisation angiogram showing obliterationof the AVM. D: Pre procedure clinical photograph showingproptosis and chemosis of the left eye. E: Post procedureclinical photograph showing full resolution at three monthsfollow up.

G.3 Pial AVM (Fig.18 & 19)Arterio-venous malformations (AVMs) are the most

common intracranial vascular malformation. AVM’s occurin about 0.02% to 0.05% of the population. In treatingAVMs, the nidus of the lesion must be removed orobliterated. The more the nidus of the AVM is occluded,the less collateral supply it will develop. The materialsavailable for embolisation include N-butyl-cyanoacrylate(NBCA) and absolute alcohol or onyx

A B CFig 18: Pial AVM

Pial AVM : 35 year old man presented with sudden onsetof hemiplegia and aphasia. CT scan revealed large rightintra cerebral hematoma. A: Carotid angiogram AP viewshows a motor cortex AVM. B: Microcatheter placed distallywithin the nidus. C: Following selective catheterizationand embolisation with NBCA, total disappearance of thenidus. Patient made a remarkable recovery.

G.4 Spinal AVM (Fig.20)In patients with spinal AVM or spinal dural fistula

embolisation is the choice of treatment as surgical excisioncarries high morbidity and mortality

A B

C DFig. 20 : SPINAL AVM

Spinal AVM. 64 year old gentleman presented with suddenonset of paraplegia. A & B: Spinal angiogram revealsspinal dural AVF in the right side of the thoracic spinalcord. C: Post embolisation shows complete occlusion ofthe AVM. D: Embolic cast.

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A B C

Fig. 19: Pial AVM – Onyx embolisation

Onyx embolisation. 20 year old medical student presentedwith recurrent seizures. CT scan revealed large AVM in theleft temporal lobe. A: Carotid angiogram shows left temporalAVM with feeders from MCA. B: Post Embolisationangiogram shows complete occlusion of the AVM.C: X-Ray skull shows onyx cast.

Absolute alcohol has the ability to penetrate the capillarywith destruction of all cells, which makes it a dangerous tool.Patients with AVM present with intractable headache,hemorrhage, seizures or progressive neurologic deficit.Cerebrovascular surgical excision of the AVM of brain is avery good option of treatment in non eloquent parts of thebrain which are located superficially. Embolisation is analternative to surgical technique in several areas that areeloquent.


Rapid advances in neuroimaging and improvementsin neuro angiography have led to better understanding ofspinal cord vascular malformation. The clinicalpresentation of spinal AVM is directly influenced by thelocation as well as the angioarchitecture. Significanttechnical improvement in catheters and delivery systemsas well as in various embolic materials have led to markedtechnical improvements in endovascular therapy.

G.5 Tracheo-bronchial stentPatients with large airway obstruction arising from

either benign or malignant processes and those withtracheo oesophageal fistula have a particularlychallenging clinical problem. Common causes of localairway obstruction include overgrowth of granulationtissue, fibrosis, tracheomalacia, endoluminal neoplasmand extrinsic neoplasm. The recent development of anendoluminal stent offers a relatively simple and noninvasive method of relieving clinical problems due toobstruction(26,27). Being a simple procedure, theplacement of endoluminal tracheo-bronchial stents hasbeen advocated for treating mechanical causes of airwaycompromise that are not amenable to surgery. After theplacement of endoluminal tracheobronchial stents,patients experience quick relief of respiratory symptomsand enjoy a relatively comfortable life. Stents alsoprovide relatively long lasting relief against malignantobstruction.

G.6 Pulmonary AV FistulaCongenital pulmonary arterio-venous fistulae (AVF)

and malformations (AVM) are abnormal directcommunications between a pulmonary artery andpulmonary vein. Blood is shunted directly from the rightheart to the left, without benefiting from two majorfunctions of the pulmonary capillary bed: oxygenationand filtration. Patients with such lesions may presentwith symptoms of hypoxia due to shunting, resistantpolycythemia, effort intolerance, high output cardiacfailure or more seriously paradoxical embolisation. Thetherapy of pulmonary AVFs is embolisation. Occlusionof the feeding artery can be accomplished with coils ordetachable balloons. Recurrence after embolisation isunusual (less than 5%).

G.7 Venous malformation / hemangioma (Fig. 21 & 22)Vascular malformations are composed of

dysplastic vessels with a normal endothelial turnover.These can be categorized into slow flow vascularmalformation (capillary, venous and lymphatic andhigh flow vascular malformation (Arterio venousvascular malformations). These venous malformationsare usually diagnosed based on clinical examinationand demonstrate phleboliths on X-ray. Therapeuticstrategies for these malformation is based on multidisciplinary approach involving dermatologists,vascular surgeons, plastic surgeons and hematologistsin addition to interventional radiologists.

A B

C DFig. 21 : Superficial Venous Malformation

Superficial Venous Malformation: 35 year old lady presentedwith multiple tortuous swelling in the right side of neck.A: Common carotid angiogram revealed no arterial feeders.B: Direct puncture embolisation was done using ethanol andlipiodol mixture. C: Pre embolisation clinical photograph withturgid neck swelling. D: Post embolisation clinical photographreveals almost marked reduction of the swelling.

Percutaneous embolisation help us to achieve significantreduction in size there by helping the surgeon to achieveadequate reduction with limited blood loss. Hemangiomasare pediatric vascular lesions which usually manifest withinthe first month of life. More than 90% show spontaneousregression at 5-7 years of age. Most hemangiomas do not requireany treatment. Embolisation is performed in those patientswho presents with high output failure to minimize the shuntand in patients who present with cosmetic disfigurement.

A B C

D EFig.22 : Upper Eyelid Hemangioma

Upper Eye lid haemangioma. 1 year old child presented withswelling of the right upper eye lid of six months duration. A:Right ICA angiogram reveals haemangioma supplied byophthalmic artery. B: Right ECA angiogram shows feeders frominternal maxillary artery. C: Post embolisation angiogram showssignificant reduction in vascularity of the haemangioma. D: Preembolisation clinical picture with mechanical ptosis. D: Postembolisation clinical picture shows remarkable reduction in sizeof the haemangioma and improvement of ptosis.

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H) INTERVENTION FOR PAIN RELIEFH.1 Vertebroplasty (Fig.23)

Vertebroplasty with acrylic glue (polymethylmethacrylate, PMMA) is a procedure aimed at preventingvertebral body collapse and relieving pain in patients withpathologic vertebral bodies / collapse due to benign ormalignant disease

A B

Fig. 23 : Vertebroplasty

Vertebroplasty. 60 year old female presented with severeback ache. X-ray revealed collapse of the L1 vertebrae.A: AP view shows acrylic glue injection with filling of theentire vertebral body. B: Lateral view showingtranspedicular approach.

The pain reducing effect of cement cannot be explainedby the consolidation of the pathologic bone alone.The PMMA is cytotoxic owing to its chemical and thermaleffect during polymerization. The temperature duringpolymerization is high enough to produce coagulationof tumoral cells. The procedure is contraindicated in patientswith hemorrhagic diathesis and in the presence of infection.An anterior approach is used in the cervical area, atranspedicular or intercosto vertebral route in the thoracicarea and a postero-lateral or transpedicular route in thelumbar area.

H.2 Percutaneous management of Osteoid OsteomaOsteoid osteoma produces local pain that is worse

at night and improves dramatically with aspirin. Effectivetreatment of this tumor depends on complete removalof the tumor nidus. The conventional treatment issurgical or percutaneous excision. The ability to preciselycontrol the treated area, a high degree of precision,applicability in joints and an excellent dose – responsecharacteristic makes interstitial laser photocoagulation(ILP) a valuable treatment method for Osteoid osteomas.It consists of percutaneous insertion of optical fibers intothe tumor. The tumor is coagulated and destroyed bydirect heating. The procedure is performed underCT guidance. A single needle and a single laser fibreare sufficient for nidus diameter upto 10mm. Successrates of 60-90% are reported. Other interventionsfor pain relief include intraarticular injection ofcorticosteroids in faget syndrome, percutaneous epiduraland nerve root block.

H.3 Percutaneous laser disc decompression (PLDD)Percutaneous removal of the nucleus pulposus has

been performed using a variety of chemical and mechanicaltechniques for the past several years. These techniquesconsist of percutaneous removal all or part of the nucleuspurposes to induce more rapid healing of the abnormallumbar disc.

H.4 Pelvic congestion syndrome (Fig.24)Chronic pelvic pain is a perplexing and disturbingly

frequent problem. An estimated 10 million women areaffected, but a reasonable explanation can be found in fewerthan half. A wide variety of gynaecologic conditions canbe responsible for chronic pelvic pain. When dilated gonadaland periuterine veins are determined to be the etiology ofthe pain, the term “pelvic congestion syndrome” is applied.Reflux of blood in gonadal veins is the underlying etiologyof pelvic varicosities in the majority of patients. Rarely,pelvic AV malformations or fistulas may be encountered.Gonadal venography remains the definitive diagnosticimaging modality. As most of the patients with pelviccongestion syndrome are relatively young, embolotherapyrepresents an attractive alternative. Coils are the embolicagents used most often for gonadal vein occlusion.Embolisation reduces or eliminates symptoms in up to 80%of women.

A B

Fig. 24 : Pelvic Congestion Syndrome

Pelvic Congestion Syndrome. 37 year old doctor presentedwith severe lower abdominal pain. Doppler revealedmultiple venous channels in the pelvis. A: Ovarianvenogram reveals multiple dilated tortuous venous channels.B: Post embolisation venogram shows complete occlusionof the dilated venous channels. Patient made a remarkablerecovery.

I) INTERVENTION FOR INFERTILITYI.1 Varicocele embolisation

Dilatation of the pampiniform plexus termed a“varicocele” results from reflux of blood throughincompetent gonadal vein valves in males. These arecommon lesions, found in 5-17% of males. There is noeffective medical treatment for varicoceles. The goal ofintervention is to interrupt the internal spermatic vein inorder to prevent retrograde flow of blood into the scrotum.Surgical ligation can be performed at multiple levels. Therecurrence rate is approximately 10-20% due to collateral

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flow. Percutaneous embolisation can be performed andembolisation with coils is preferred. Multiple coils aredeposited along the entire length of the vein. Sperm countsimprove in 80% of patients following successfulembolisation

I.2 Vasculogenic impotenceThe indications for penile angiography are the

evaluation of impotence and trauma. Approximately 50%of males over the age of 40 experience some degree oferectile dysfunction. The least common cause isvasculogenic, and should be pursued only after otheretiologies have been excluded. There are two potentialvascular causes of impotence, venous leak (inability totrap blood in the corpus cavernosum) and arterialinsufficiency. Vasculogenic impotence has a venousetiology in one third of cases and combined arterial andvenous in the rest. Patients with venous leak tend torespond well to pharmacologic therapies. Severe arterialinsufficiency is more difficult to treat effectively but in aninternal iliac artery, angioplasty or microvascular bypassto the penis may be effective.

I.3 Uterine artery embolisation (Fig. 25)The most common indication for selective uterine

artery angiography is for embolisation of symptomaticfibroids. Fibroids are vascular tumors that grow in sizeand increase in prevalence with age throughout a woman’sreproductive life. The indications for intervention arefibroids that cause heavy, prolonged periods, pelvic pain,dyspareunia, miscarriages and pressure symptoms onadjacent structures. Pharmacologic treatment with GnRHanalogues, results in temporary reduction in size, butfibroids will enlarge once medication is stopped.Conventional surgical procedures include hysterectomy andmyomectomy, using open, laparoscopic or hysteroscopictechniques. Uterine artery embolisation is an alternativeapproach to management of fibroids

A BFig. 25: Uterine Fibroid Embolisation

Uterine Fibroid Embolisation. 35 year old lady presentedwith abnormal uterine bleeding. Ultrasound revealed 6 x 4cms fibroid in the right anterior wall of uterus. A: Selectiveuterine artery angiogram reveals hypertrophied feeders tothe fibroid. B: Post embolisation angiogram revealssignificant reduction of flow and disappearance of theabnormal vascularity.

The basic principle is selective infarction of thefibroids with particulate embolic materials directly

delivered into the uterine arteries. The indications areidentical to those for surgery, although the procedure isnot recommended when the fibroids are pedunculated orlargely submucosal. Permanent particles (300-700mmdiameter range) are generally used. Embolisation iscontinued until there is sluggish flow in the uterine arterywith elimination of the fibroid blush. Bilateralembolisation is mandatory. Embolised fibroids shrink onaverage 50-60% in volume, with relief of symptoms in85-90% of patients.

I.4 Fallopian Tube Recanalisation (Fig.26)Isolated obstruction of the proximal fallopian tube

amenable to transvaginal dilatation and recanalisation isthought to be the cause of infertility in about 20% of thepatients.

A B

Fig.26: Fallopian Tube Recanalisation

Fallopian Tube Recanalisation. Patient presented withinfertility following right salpingectomy for right tubalpregnancy. A: HSG showing left cornual block. B: Balloondilatation of the left cornu done, contrast injection showingperitoneal spillage

Women with unilateral or bilateral proximal tubalobstruction confirmed by hysterosalphingography orlaparoscopy are candidates for transvaginal recanalisation.Transvaginal fallopian tube recanalisation results in lowerpatient morbidity and is less expensive than tubalmicrosurgery. This procedure is recommended as theintervention of first choice in patients with proximal tubalobstruction. The more invasive therapy should be reservedfor patients with distal tubal disease and for those in whomfluoroscopic catheterization fails.

J) INTERVENTION FOR PALLIATIONJ.1 Percutaneous Transhepatic Biliary Drainage

(PTBD) (Fig. 27)The major purpose of PTBD has been and is still

used to drain retained bile for decompression inobstructive jaundice. PTBD route is indispensable forcarrying out various procedures of biliary interventionalradiology. Another use of the route is to obtain an accessfor intracavitary radiotherapy. (28) Cholestasis inducesbiliary infection and protracted hyperbilirubinemiaimpairs renal function. In the presence ofhyperbilirubinemia, both surgical mortality andmorbidity are high at 15 to 25% and 40 to 60%(29,30) and

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decompression should be done as early as possible inpatients with obstructive jaundice. The success rate ofPTBD is 90-100%. (31,32) The application of indwellingstents (endoprostheses) in the biliary system has been wellestablished because it provides sufficient antegrade biliarydrainage without encumbrance of an external tube thatneeds catheter flushing, dressing at the skin entry siteand periodic catheter exchanges.

A B C

Fig. 27: Percutaneous Transhepatic Biliary DrainagePercutaneous Transhepatic Biliary Drainage. 70 year oldmale presented with obstructive jaundice. MRI revealedKlatskin tumor. A: Cholangiogram shows obstruction atthe level of hepatic duct confluence. B: Guide wire placedin the duodenum across the stricture. C: Followingsuccessful stent placement, flow of contrast through theobstructed segment visualized. Patient experienced markedrelief following biliary stenting.

Invention of metallic stents has become a popularprocedure of palliative treatment for patients with obstructivejaundice caused by unresectable malignant tumors. Thelarge caliber is associated with a low frequency of occlusionby bile encrustation.

J.2 Tumor ablation (Fig. 28)Many solid tissue malignancies are poorly responsive

to systemic chemotherapy, surgical resection or localradiation therapy. In situ image guided tumor destructionor ablation has become an attractive option. It offers thepossibility of an effective minimally invasive and lesscostly approach, often achievable in an outpatient sitting.Available ablation techniques can be broadly classified aschemical, embolic or thermal. Chemical ablation isachieved by image – guided instillation of a chemical agent.The most common chemical agent used for tumor ablationis ethanol. Percutaneous ethanol injection (PEI) has beenshown to be a safe, inexpensive and effective treatmentfor small (3-5cms) hepatocellular carcinomas. Tissuefunctions normally in a narrow range of temperatures. Ifthe local temperature is made sufficiently abnormal, thecells within the environment are permanently damaged.If extremes of temperature are applied, the cells aredestroyed and coagulative necrosis ensues. Percutaneous,image – guided therapies using heat have utilized diversethermal energy sources. Thermal energy sources haveincluded sound (high – intensity focused ultrasound, light(laser photocoagulation), microwaves and radio frequencyenergy.

A B C

Fig.28: RadioFrequency AblationRadiofrequency Ablation. 58 year old male known case ofadenocarcinoma lung underwent RFA. A: CT Thorax revealsa large tumor in the posterior segment of the right upperlobe. B: Three months follow up reveals cavitation of thetumor with marked reduction of size. C: Six months followup reveals almost disappearance of the tumor following RFA.Radio frequency energy has been used for surgicalelectrocautery since the early 1990s. RFA can be achievedwith monopolar or bipolar electrode systems.

COMPLICATIONSComplication related to interventional radiological

procedures include puncture site related, contrast agentrelated, catheterization related and those related tocoagulation. Puncture site related complications includepuncture site hematoma, arterial dissection, pseudo aneurysmformation and arterio venous fistula. Contrast agents maylead to nephropathy, allergic reaction, congestive cardiacfailure. The use of iso osmolar, non ionic contrast mediahas reduced the incidence of these complications. Injuryto vessels may occur during catheterization and is morecommon in elderly patients due to pre existentatherosclerosis and in patients with collagen vasculardiseases. Thrombo embolic complications may occur dueto dislodgement of atheromatous plaques and due toformation of platelet thrombi at the tip of the catheter. Theincidence of these complications is variable depending onthe procedure performed and the disease for which it isperformed.

ConcludedACKNOWLEDGEMENT :

Authors wish to express their thanks to Prof. RoySantosham and Dr. K. Murali for using their material forpublication.

REFERENCES:

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5) Hollier LH, Reigel MM, Kazmier FE, et al.Conventional repair of abdominal aortic aneurysm in thehigh – risk patient: a plea for abandonment of nonresectivetreatment. J Vasc Surg 1986; 3:712-717.

6) The STILE investigators. Results of a prospectiverandomized trial evaluating surgery versus thrombolysis forischemia of the lower extremity. Ann Surg 1994; 220:251-268.

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9) Muthuswamy PP, Akbik F, Franklin C, et al.Management of major or massive hemoptysis in activepulmonary tuberculosis by bronchial arterial embolisation.Chest 1987; 92: 77-82.

10) UFlacker R, Kaemmerer A, Neves C, et al. Managementof massive hemoptysis by bronchial artery embolization.Radiology 1983; 146:627-634.

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18) Heiss WD, Graf R, Weinhard J, et al. Dynamicpenumbra demonstrated by sequential multitracer PET aftermiddle cerebral artery occlusion in cats. J Cereb BloodFlow Metab 1994; 14:892-902.

19) Jellinger K. Pathology and aetiology of intracranialaneurysms. In Pia HW, Langmaid C, Zierski J (eds).Cerebral Aneurysms in Diagnosis and Therapy. New York;Springer, 1979, pp. 5-19.

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21) Stehbens WE. Aneurysm and anatomical variation ofthe cerebral arteries. Arch Pathol 1963; 75:45-64.

22) Adams HP Jr, Kassell NF, Torner JC, et al. Earlymanagement of aneurysmal subarachnoid hemorrhage; areport of the cooperative aneurysm study. J Neurosurg 1981;54:141-145.

23) Kassell NF, Torner JC, haley EC, et al. TheInternational Cooperative Study on the Timing of AneurysmSurgery. Part 1: Overall management results. J Neurosurg1990; 73:37-47.

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27) Varela A, Maynar M, Irving D al. Use of Gianturcoself-expandable stents in the tracheobronchial tree. AnnThorac Surg 1990; 49: 806-809.

28) Karani J, Fletcher M, Brinkely D, et al. Internal biliarydrainage and local radiotherapy with iridium – 192 wire intreatment of hilar cholangiocarcinoma. Clin Radiol 1985;36:603-606.

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Dr. ANUPMA JYOTI KINDOAssociate ProfessorDepartment of MicrobiologySri Ramachandra Medical College and Research InstitutePorur, Chennai - 600 116email : [email protected] Dept. of Microbiologyb Dept. of Dermatology

CLINICO – MYCOLOGICAL PROFILE OF DERMATOPHYTIC SKIN INFECTIONS IN ATERTIARY CARE CENTER – A CROSS SECTIONAL STUDY.Kennedy Kumar a, Anupma Jyoti Kindo a, J. Kalyani a, S. Anandan b

INTRODUCTIONMycotic infections are world wide in distribution.

However, superficial mycosis is more prevalent in tropicaland subtropical countries including India, where heat andmoisture play an important role in promoting the growth ofthese fungi [1, 2].

Fungal infections have attracted the attention ofphysicians and microbiologists in recent years due to variousreasons like indiscriminate use of antibiotics, anticancertherapy and immunodeficient diseases like AIDS.

Sex, race and occupation have little recognizeddifferential influence upon the frequency of dermatophytosis[3], however change in trends are noticed in the studiesdone by the later researchers.

In the current study, we have undertaken a clinico-mycological approach, correlating various demographic datasuch as age, and sex with identification of the fungus usingstandard techniques [3]. As the dermatophytic skin infectionsare more frequent when compared to those of hair and nailsthe study was confined to skin infections alone.

Materials and methodsA total of 117 consecutive patients with skin lesions

resembling tinea attending the Dermatology Out PatientDepartment of Sri Ramachandra Medical College and

ABSTRACTIntroduction: The prevalence of dermatophytosis differs fromplace to place and is governed by environmental conditions,personal hygiene and individual’s susceptibility.Aim and objective: This study is sought to determine theprevalence of dermatophytic skin infections and theircausative agent in the population attending the DermatologyOut patient department.Material and methods: A total of 117 patients with skinlesions resembling tinea infections attending out patientdept during a six month period (February 2004 to August2004) were taken for study. Diagnosis was confirmed bymicroscopy and culture.Results: Out of the 122 samples collected from 117 patientsdermatophytes were isolated from 54.9 %,( 67isolates) non-

dermatophytes 6.6% (8 isolates) and candida from 4.1% (5isolates). Tinea corporis accounted for 70.8 %( 82 cases)followed by tinea cruris 18.8% (22 cases). Tinea faceiiand tinea manuum 3.4% (4cases) each and mixed infectionin 5 patients. The male to female ratio of the skin infectionwas 1.12:1 T. rubrum was the most common etiologicalagent in 45 cases (67.5%). T. mentagrophytes 18.0% (12cases), other dermatophytes isolated were T. schoenleinii4 , two each of E. flocossum and M.audouinii and one ofM. nanum and M. canis.Conclusion: Among the dermatophytic skin infections tineacorporis was the predominant clinical type and T.rubrumwas the most common dermatophyte isolated.

Key words: Cutaneous fungus, dermatophytes, Tinea

Research Institute, Porur, Chennai, Tamilnadu were takenfor the study over a period of six months (February 2004 toAugust 2004).

A detailed history of selected cases was taken in relationto name, age, sex, address, occupation, duration of illnessand involvement of more than one site.

Samples were collected from the site of the lesion.Scrapings were taken with a blunt sterilized scalpel fromthe active site of the lesion using standard techniquedescribed earlier [3].

All the samples collected were subjected to microscopyand culture.

Following direct microscopic examination with 10%KOH, the scrapings were inoculated into slopes of duplicatesets of tubes containinga) Sabouraud’s dextrose agar with chloramphenicolb) Sabouraud’s dextrose agar with thiaminec) Sabouraud’s dextrose agar with chloramphenicol and

cycloheximide (To prevent contamination withsaprophytic fungi and bacteria.)

d) Dermatophyte test medium.

One set of the tube was incubated at 37°C and theother set at 25°C. The cultures were examined every twodays for a period of one month for the presence of growth.The growth was observed starting from sixth day onwards.If no growth was found after 45 days it was considerednegative for the growth of fungi.

Growths obtained were identified based on the colonymorphology, microscopic appearance and other relevant testsas described by Emmons. The growth on the tubes wasexamined for gross morphology, pigmentation if any andslide cultures were put to identify up to species level.

Original Article


ResultsA total of 11847 patients attended the OPD section of

Dermatology, Venereology and Leprology during the periodof our study. Hundred and seventeen of them fulfilled thecriteria (not taken any treatment and also had infection forthe first time) and hence taken up for the study. There were62 men (53%) and 55(47%) women. The age of the patientsranged from 4-83 years, the mean age being 35.8 years.There was preponderance of males in the 11-30 years andfemales in the age group of 31-50 years. Very few caseswere encountered in the extremes of age. Males weremarginally more affected than females, male to female ratiobeing 1.12:1. The sex distribution of various clinical typesis depicted in Table1.Tinea corporis was the predominantlesion in the present study occurring in 82 (70.08%) patientsfollowed by tinea cruris in 22 (18.8%), tinea faceii andtinea manuum each in 4 (3.41%) and mixed infection in5 (4.27%) patients.

Table-1SEX DISTRIBUTION OF

DIFFERENT CLINICAL TYPES OF DERMATOPHYTESClinical types Male Female Total

T.corporis 32 50 82

T.cruris 20 2 22

T.manum 3 1 4

T.facieii 2 2 4

Mixed 5 0 5

Total 62 55 117

Tinea corporis was found to be the most commonclinical presentation.

The mixed infection included a combination of tineacorporis and tinea cruris in three patients, tinea corporisand tinea capitis in one and tinea faceii and tinea manuumin the other. The dermatophytic infections were morecommon in the third decade.

Dermatophytes formed the majority accounting for67 out of the total 122 samples (54.9%). The principaldermatophyte was T.rubrum 45(67.5%) followed byT.mentagrophytes 12(18.0%), 4 of T.schoenleinii 2 each ofE.floccosum and M.audouinii and 1 of M.nanum andM.canis.

A single species of dermatophyte could cause differentclinical manifestation as seen with T. rubrum being themajor isolate, from all the clinical types of tinea; and asingle clinical type like tinea corporis had several etiologicalagents, from all the three genera of dermatophytes namelyTrichophyton, Epidermophyton and Microsporum.(Table 2)

Non-dermatophytic molds were grown from 8 (6.6%)and candida from 5 samples (4.1%). The various non-dermatophytic molds isolated were as follows: Exophialaspp two in number and one each of Exserohilum spp.,Fusarium spp.,

Original Article

TABLE 2CO-RELATION BETWEEN CLINICAL AND

MYCOLOGICAL TYPES OF DERMATOPHYTOSISDermatophytes Tinea Tinea Tinea Tinea Mixed Totalisolated corporis cruris manum faceiiT.rubrum 21 13 3 2 6 45

T.mentagrophytes 6 4 0 2 0 12

T.schoenleinii 4 0 0 0 0 4

E.floccosum 2 0 0 0 0 2

M.audounnii 1 1 0 0 0 2

M.nanum 1 0 0 0 0 1

M.canis 1 0 0 0 0 1

Nigrospora spp., Scopulariopsis spp., Cladosporiumspp. and Acremonium spp. All these non-dermatophyteswere isolated in pure culture and from KOH positive samplesand no dermatophytes were isolated along with them.

In our study culture was negative in 42 samples (34.42%).

DISCUSSION:As stated earlier the present study focused only on skin

lesions caused by dermatophytes. Earlier studies confirm thatdermatophytic skin infection were more common in malesthan females as reported by Bhaskaran et al. from Tirupatiand Maheshwari Amma et al [4,5], the ratio being 2:1.While most studies in and around Chennai showed a maledominance. In contrast, one study reported a femalepreponderance (67.26%) (Kamalam.A Thambiah et al) [6]In our study among the 117 patients who were clinicallydiagnosed as dermatophytosis the percentage of males (53%)was only marginally higher than the females (47%) with themale female ratio 1.12:1, which can be attributed to theincreased health awareness among the women and theirpositive attitude towards treatment without inhibition andtheir increased cosmetic consciousness.

The present study has revealed that the majority (46.2%)of the infection by dermatophytes has occurred during the3rd and 4th decades of their life, an observation which is inpar with those of the earlier studies [7,8]. The probablereason for this age predilection is excessive sweating due toexcessive physical activity, as a consequence, in additionthe tropical climatic conditions.

Tinea corporis was diagnosed in 82 (70.08%) ofthe 117 patients with dermatophytosis of the skin (fig-1).

Fig-1 : Tinea corporis showing lesion with well defined margins


Our results are comparable to those from other places likeKashmir, Jabalpur and Manipal[9,10,11]. Tinea corporishad been reported to be the most common clinical typeeven in few other countries like Spain and Brazil [12,13].

Tinea cruris (18.8%) followed tinea corporis as the nextmost common clinical variety in our study (fig-2). This reportsubstantiates that published by other authors [7].

of the Indian studies on dermatophytosis in India as wellas the Western countries. T.rubrum was the most commonisolate (56.25%) which is comparable with the reportsof other authors [7] and was associated more withtinea corporis (46.6%) than with tinea cruris (16.25%).(Fig-3)

It was found from our study that T.mentagrophyteshad an occurrence of 17.8% amongst the dermatophytes(n=67). Therefore it occupied the second place withregard to the frequency with which it was isolated, whichis comparable to other studies from India and abroadduring the last 50- 60 years, only the percentage of isolatesdiffered (ranging from 11-17%). We also found that 50%of T.mentagrophytes was from tinea corporis and 33.3%from tinea cruris. Only two cases (16.7%) were from tineafaceii.

Out of the total dermatophytes isolated it was foundthat 5.9% of them were T.schoenleinii. This particularspecies of trichophyton is rarely reported in literature.T.schoenleinii has more often been an isolate from cases offavus. Hence this is a new observation pertaining to thecurrent study and has to be ascertained by further extensivestudies covering a larger population in the same area overan extended period of time.

E.floccosum formed only 2.98% of the totaldermatophytes (n=67) in our study. Only few studies fromIndia and abroad have shown E.floccosum as one of thedermatophyte isolated [5,16].

Pure growth of non-dermatophytic molds were isolatedon repeated cultures from 8(6.6%) of the total samples..There were five isolates of Candida spp., (4.1%).

As with the reports of Wg Cdr Sanjiv Grover, Lt Col PRoy [17] this is a striking finding in our study. Thoughcommonly considered as contaminants, they have beenreported to colonize damaged tissues and cause secondarytissue destruction. Their role in causing cutaneous infectionsis not yet proven and a primary pathogenic role of non-dermatophytes is controversial at best [17]. But the fact thatthese non dermatophytic molds were isolated on repeatedcultures, without association of dermatophytes and onlyfrom KOH positive samples, bears some significance onetiology. The overall findings suggest that our studies arewell comparable to the studies conducted by otherresearchers and the pattern of dermatophytes prevalent inour subjects under study is similar to that in other parts ofthe country.

REFERENCES:1. AminAG and Shah CF: Anaylasis of 141 cases ofdermatophytoses. Indian J Dermatol Venereol Leprol 1971;37: 123

2. Desai SC and Bhatt MLA: Dermatomycosis in Bombay.Indian J Med Res 1961; 42: 662.

3. Emmons CW, Binford CH, Utz JP, and KwonchungKJ: Medical Mycology;3rd edn, Philadelpia: Lea & Febiger1977; 120-121.

Original Article

Mixed infection of tinea cruris and tinea corporis wereobserved only in negligible numbers and so also was tineamanuum, tinea capitis and tinea faceii.

The high incidence of tinea corporis and tinea cruris asconcluded from our study is probably due to its symptomaticnature (pruritis) which leads the patient to seek medicaladvice [7].

Tinea corporis was prevalent more in females (42.7%)than males (30.8%) and tinea cruris affected males (19.6%)more than females (1.7%). The reason for the higherincidence of tinea corporis is probably due to the type ofattire, favours dermatophytic infections to thrive [14].

Tinea cruris is much more common in men than inwomen. The reason for this preference may be becausemen wear more occlusive clothing and are more physicallyactive. Moreover they are at a greater risk of acquiringinfection at other sites (e.g. tinea pedis) due to their natureof work. This may act as a reservoir for new cases of tineacruris [15].

Trichophyton species have been isolated withincreasing frequency as compared to Microsporum andEpidermophyton species. This has been noticed by many

Fig-2 : Tinea cruris showing hyperpigmentatedlesion in the genital area

Fig-3 : Microphotograph of Trichophyton rubrumShowing abundant tear drop microconidia and occasional

Macroconidia (LPCB mount magnificationX200)


4. Bhaskaran CS, Rao PS, Krishnamoorthy T, TarachandP: Dermatophytoses in Tirupathi. Indian J Pathol Microbiol1977; 31: 251- 59.

5. Maheshwariamma SM, Paniker CKJ, GopinathanT.Studies on dermatomycosis in Calicut. Indian J PatholMicrobiol 1982; 25: 11-17.

6. Kamalam A, Thambiah AS. A study of 3891 cases ofmycoses in the tropics Sabouradia 1976 ;14:129-48.

7. Verenkar MP, Pinto MJW, Rodrigues S, RoqueWP,SinghI. Clinico-Microbiological study of dermatophytoses.Indian J Pathol Microbiol 1991; 186- 192.

8. SenthamilSelvi G, Kamalam A, Thambiah AS. Scenarioof chronic dermatophytosis. Mycopathologia 1998; 140:129-135.

9. Bhardwaj G, Dermatophytoses in Kashmir (India).Mycosen 1987;30:135.

10. Stephen S, Rao KNA. Superficial mycoses in Manipal,Indian J Dermatol Venereol Leprol 1975; 41:106.

11. Phadke SN . Dermatophytosis in Jabalpur (MadhyaPradesh). Indian J Pathol Bacteriol 1973;16:42.

Original Article

12. Fortuno B, Torres L, Simil E, Seoane A, Uriel JA,Santacruz C. Dermatophytes isolated in our clinics,5-year study in Zaragoza. Enferm Infecc Microbiol Clin.1997 ; 15: 536-9.

13. Chinelli PA, Sofiatti Ade A, Nunes RS, Martins JE.Dermatophyte agents in the city of Sao Paulo, from 1992to 2002. Rev Inst Med Trop Sao Paulo 2003; 45: 259-63.

14. Patwardhan N, Dave R. Dermatomycosis in andaround Aurangabad. Indian J Pathol Microbiol 1999;42: 455-462.

15. Kanwar AJ, Mamta, Chander J. Superficial fungalinfections. In: Valia RG, Valia AR, editors. IADVL textbookand atlas of dermatology. 2nd ed. Mumbai: BhalaniPublishing House; 2001. 215-58.

16. Falahati M., AkhlaghiL, Lari AR, Alaghehbandon R.Epidemiology of dermatophytoses in an area south ofTehran, Iran. Mycopathologia 2003; 156: 279-87.

17. Wg Cdr Grover S, Lt Col Roy P. Clinico-mycologicalprofile of superficial mycosis in a hospital in North-EastIndia. MJAFI 2003; 59:114-116.


LABELLING TRANSFORMED MITOTIC CELLS WITH FLUORESCENT ANTIBODY CONJUGATE

Maddaly Ravi a, Deepa Parvathi. V a, Govind Pai. M a, Preetha. B a, Sulogna Ghosh a and Solomon F.D Paul a

ABSTRACTThe functions of most living cells are characterized

by well-governed events that encompass the cell cycle.Cell division primarily occurs as mitosis, which is regulatedand follows a defined series of mechanisms. A key playerin regulating mitosis is a group of proteins collectivelycalled the mitotic factors, which are initially localized inthe cytoplasm. These appear gradually and peak at themitotic stage of cell cycle. Protein profile analysis ofinterphase and mitotic cells show distinct differences andthis was utilized for the premature condensation ofinterphase DNA through techniques involving somatic cellhybridizations and mitotic extracts. Studies of cell cyclekinetics and the underlying mechanisms have becomepossible with the continuous cultures of cell lines invitro. We have raised polyclonal antibodies to the mitoticproteins of Chinese Hamster Ovary cells and the


Dr. MADDALY RAVIAssistant Professor, Department of Human Genetics,Sri Ramachandra Medical College and Research InstitutePorur, Chennai – 600 116.Email : [email protected] of Human Genetics

INTRODUCTION:Cell growth and division comprises a series of discrete

stages, collectively known as the cell cycle (1). The cellcycle begins when the division of a single parental cellforms two new cells and ends when one of these cells dividesagain into two cells. This division process, called the “Mphase”, involves two overlapping events, the karyokinesisfollowed by cytokinesis (2). Although the cells of amulticellular organism divide at varying rates, most studiesof the cell cycle involve cells growing in culture where thelength of the cycle tends to be similar for different celltypes. Determining the overall length of the cell cycle - thegeneration time – for cultured cells has been achieved andreported earlier (3). For mammalian cells in culture, S phaseis about 6- 8 hours in length. Similarly, estimating the lengthof M phase by multiplying the generation time by thepercentage of the cells that are actually in mitosis at anygiven time is possible. This percentage is called the Mitoticindex. The mitotic index for cultured mammalian cells isoften about 3-5 %, which means that M phase lasts lessthan an hour (usually 30 -45 minutes) (4).

The cell cycle control system is based on two keyfamilies of proteins. The first is the family of cyclindependent protein kinases (Cdk), which induce downstreamprocesses by phosphorylating selected proteins on serinesand threonines. The second is a family of specialized

predominantly IgG antibodies from the whole serum werepurified following induction of a secondary immuneresponse. The purified IgG was conjugated to FITC, afluorescent dye and the conjugate was used to staininterphase and mitotic cells of immortalized cells in cultureand also cultured peripheral blood human lymphocytes. Itwas observed that the conjugate selectively stainedimmortalized mitotic cells either in suspensions or inpreserved tissue sections. While the cross reactivity withimmortalized mitotic cells of varied types was evident,the non-reactivity to untransformed cells gives us a meansof further utilizing these antibodies to potentialapplications such as in cancerous conditions where celltransformation and uncontrolled mitotic events are theunderlying factors.

Key words: Chinese hamster ovary cell line, polyclonalantibodies, mitotic factors, immunofluorescence.

activating proteins, called cyclins that bind to Cdk moleculesand control their ability to phosphorylate appropriate targetproteins. The cyclin assembly, activation and disassemblyof cyclin-Cdk complexes are the pivotal events driving thecell cycle (3). Cyclins undergo a cycle of synthesis anddegradation in each division of the cycle of the cell. Thereare two main classes of cyclins: mitotic cyclins, which bindto Cdk molecules during G2 and are required for entry intomitosis, and G1 cyclins, which bind to Cdk moleculesduring G1 and are required for entry into S phase.

Cell fusion experiments suggested that specificmolecules present in the cytoplasm are responsible formoving the cells through the G1 and G2 checkpoints (thatis, for triggering the onset of DNA replications phase) andmitosis (M phase) (5). Evidence regarding the mitosis-triggering signal has come from experiments involving frogeggs. During development of the frog oocyte (an egg cellprecursor) the cell cycle is arrested in G2 until hormonesstimulate meiosis. The oocyte then proceeds through mostof the phases of meiosis but is arrested during metaphase ofthe second of two meiotic divisions. If cytoplasm takenfrom a mature egg cell is injected into the cytoplasm of animmature oocyte, the oocyte immediately begins meiosis(6). It was hypothesized that a cytoplasmic molecule, whichwere named maturation promoting factor (MPF), inducesthis oocyte “maturation”.

Subsequent experiments revealed that in addition toinducing meiosis, MPF can also trigger mitosis when injectedinto fertilized frog eggs (6). Similar MPF molecules werelater discovered in the cytoplasms of a broad range of dividingcell types, including yeast, marine invertebrates, andmammals. Because of the general role played by MPF intriggering passage through the G2 checkpoint and intomitosis, MPF, which originally stood for “maturationpromoting factor” is now used to mean “mitosis promotingfactor”, a term that describes this molecule’s role.

Original Article


As distinct protein profiles are evident in the interphaseand mitotic phases of the cell cycle, we utilized the antigenicproperties of proteins present in the mitotic stage to raisepolyclonal antibodies against the same. Antibodies beingvery specific in their nature of activity, we attempt todemonstrate the specific reactivity of these antibodies tocells at the mitotic stage of transformed cells.Immunofluorescent techniques provide us with the bestpossible visualization in cytological applications andtherefore we used FITC conjugated antibodies todemonstrate the localization and reactivity to varied celltypes in both interphase and the mitotic stages.

MATERIALS AND METHODS:

Polyclonal antibodies to CHO mitotic cytosolic proteins:A continuous culture of CHO cell line was maintained

according to standard protocol. Briefly, the cultures weremaintained in DMEM with 5% serum supplementation at370 C under 5% humidified atmosphere (Fig1).

to obtain a final titre of 1:8 and was used for the FITCconjugation. The chromatogram of IgG affinity purificationis given in Fig 2.

FITC conjugation to Anti-CHO mitotic protein antibodies

Figure 1: CHO cells in culture. Typically, cells show attachmentin a few hours after passaging and attain healthy confluence inabout 48 hours in a T-25 flask. Culture conditions are in DMEMsupplemented with 5% FBS; 370 C with 5% CO2.

Mitotic CHO cells were harvested and the cytosolicproteins extracted as previously described. Rabbit polyclonalantibodies were raised against the extracted mitotic cytosolicproteins and identified IgG as the predominant isotypefurther to the induction of secondary immune response (7).IgG was affinity purified from whole serum, concentrated

Fluorescein isothiocyanate (1.0 mg) was reconstitutedwith 200µL of the solvent (sterile water or 40-50% glycerol).250 µL of affinity purified IgG fraction (after concentration)was mixed with 1/10th volume (25 µL) of sodium carbonatebi carbonate buffer [7.5 mL of 0.2 mol/L sodium carbonatesolution diluted with 42.5 mL of 0.2 mol/L sodiumbicarbonate solution to 200 mL with distilled water]. 15 µLof reconstituted FITC solution was added to the above andincubated at room temperature for 2 hours on a shaker indark. Following incubation, 1/20th volume (30 µL) of 1Mammonium chloride was added and incubated at roomtemperature for 1 hour on a shaker in dark. Sephadex G-25column was washed with 20 mL 1X PBS to obtain base lineUV and conductivity levels. 300µL of reaction mixture (IgGlabeled with FITC) was loaded to the bed of the columnand eluted through the column using 1X PBS as the washbuffer. The elution profile was recorded and the first fraction(containing the IgG-FITC conjugate) was collected. The IgG-FITC conjugate was stored at 4°C with 1%(w/v) BSA and0.1% sodium azide.

Target cell preparation and immunostaining:While interphase cells of the cell lines in culture

(CHO, Vero and HeLa) remain attached to the culture flasks,those in the mitotic phase round off and detach from theculture flasks (Fig 3). Mitotic cells were collected from celllines by gentle mechanical agitation from confluent flasksand were individually processed by washing (1000 rpm;10minutes) thrice in 1X PBS.

A 72-hour human lymphocyte (whole blood) culturewas set up according to conventional cytogenetic

Figure 2: Chromatogram of IgG purification from rabbit antiCHO mitotic cytosolic protein whole serum. Further to thesecondary booster, the predominant antibody Isotype was IgGas determined by Ouchterlony Double Diffusion. IgG waspurified to avoid the usage of whole serum to minimize non-specific interactions.

Original Article


procedures. The culture was initiated in RPMI 1640medium with 10%FCS and incubated at 37°C, 5% CO2.Phytohaemagglutinin was added to stimulate lymphocyteproliferation in vitro.100µL of 0.01% Colchicine(mitotic blocker) was added at the end of 70 hours andincubated for 90 minutes; following which the cells wereharvested by a brief exposure (20 minutes) to hypotonicsolution (0.075M pre warmed Potassium Chloride),centrifuged at 1000 rpm for 10 minutes and the cell pelletwas collected.

The cell pellets thus collected of the various cell typesemployed for this study were incubated in 150 µL blockingbuffer for 30 minutes at room temperature and followingincubation they were washed twice in 1X PBS and the cellpellets were resuspended in minimum quantity of 1X PBS.To each of the cell pellets 200 µl of IgG-FITC conjugatewas added and incubated at 4°C overnight. Followingovernight incubation the cells were washed (1000 rpm; 10minutes) in 1X PBS and layered on glass slides and observedunder the fluorescence microscope (Flow chart 1).

Figure 3: CHO Interphase and Mitotic cells; upon trypsinization,cells detach from culture flask enabling passaging andmaintenance of the cell line. In a healthy culture, Interphasecells remain attached to the flask with distinct spindle shapedmorphology and the Mitotic cells round-off and detach fromthe flask surface; thus enabling selective harvest of cells at theMitotic stage.

Flowchart 1: Protocols employed for obtaining targetcells and their processing for immunofluorescent staining.

Figure 4: Immunofluorescent staining of epithelial tissue sectionby the Anti CHO mitotic cytosolic proteins IgG-FITC conjugaterevealing selective staining of cells.

Epithelial tissue that was paraffin embedded andpreserved was washed in 1X PBS for 5 minutes three times.The section was rehydrated in alcohol series (95%, 90%,85% ethanol) for 2 minutes in each three times. The slide

Original Article


was rinsed in 1X PBS for 5 minutes twice. The section wasincubated in blocking serum for 15 minutes. Followingincubation 250µL of IgG-FITC conjugate was added to itand the slide was incubated at 4°C overnight in a humidchamber. Following overnight incubation, the slide wasrinsed once with 1X PBS and observed under the fluorescencemicroscope.

RESULTS:Concentrated IgG fraction (250µL) was conjugated with

FITC and purified by desalting to remove unbound dye fromthe conjugate. A final volume of 2 ml of the purified IgG-FITC conjugate was collected.

A mixture of interphase and mitotic cells from CHO,Vero, HeLa and human peripheral blood lymphocytesharvested and stained with IgG –FITC conjugatedemonstrated selective staining of mitotic cells. The samewas observed on paraffin embedded epithelial tissue sections;only a few cells in the tissue were preferentially stained bythe IgG-FITC conjugate (Fig 4).

DISCUSSION:Mitotic cells of human origin (HeLa), upon fusion,

can induce Premature Chromosome Condensation in cellsfrom a variety of animal species including mammals, birds,amphibians, fishes, and insects, and mitotic cells from thesespecies can induce PCC in HeLa cells indicates that thefactors involved in the induction of this phenomenon arecommon over a wide range of animal species. In fact plantcells have been shown to induce PCC in mammalian cells.The factors involved are most likely proteins since, if themitotic inducer cells are prelabeled with radioactively taggedaminoacids, labeled protein can be observed to be transferredfrom the mitotic component to the interphase chromosomes(8, 9).

The mitotic factors being proteins biochemically, theirantigenic properties can be fairly well exploited and suitableantibodies produced that can specifically react with thesame. Such antibodies, moreover if polyclonal in nature, byvirtue of the affinity to antigenic epitopes can be usefultools for evaluating homogeneity of mitotic cytosolic proteinantigens. Also, the cross reactivity of the antibodies tostructurally similar antigenic epitopes might prove to bevaluable in comparing soluble proteins. Immunofluorescentand immunohistochemical techniques employing suitableantibody conjugates become useful detection tools forspecific cell populations or those that express uniqueantigenic epitopes.

The cross reactivity of polyclonal rabbit Anti CHOmitotic extract with mitotic cells of CHO, Vero, HeLa andnormal human lymphocytes was earlier demonstrated toevaluate the homogeneity of the cytosolic protein antigens(10). The results demonstrated the reactivity of the wholeserum and IgG fraction only to the mitotic extracts fromimmortalized cell lines (CHO, Vero and HeLa) and failed

to react with the protein extracts from normal humanlymphocytes indicating the presence of unique protein orantigenic epitope in mitotic extracts of cell lines with alteredcell cycle kinetics which is not present in normal cells(human lymphocytes). It was also inferred that the reactivityof the antiserum is strongly influenced by the presence ofthis particular protein or the epitope.

CONCLUSION:The fluorescent antibody conjugate employed in this

study was effective in detection and selective labeling ofimmortalized mitotic cells from a mixed population eitherin suspension or in preserved tissue section. This whiledemonstrating the presence of shared epitopes in alteredcell cycle conditions, also gives us the possibility ofexploitation of these antibodies for a variety of applicationsespecially in cancerous situations where, the breakdown ofthe mitotic machinery is the hall mark that results inunregulated mitotic proliferation of cells.

REFERENCES:1) Becker WM, Kleinsmith LJ, Hardin J, The world ofthe cell. 4th ed. San Francisco: The Benjamin/CummingsPublishing Company; 2000, 533-581.2) Cooper GM, Hausman RE, The Cell: a molecularapproach. 3rd ed. Washington, D.C: ASM Press 2004, 591-625.3) Alberts B, Bray D, Lewis J et al., Molecular biology ofthe cell.3rd ed. New York: Garland Publishing 1994, 863-943.4) Lodish H, Baltimore D, Berk A et al., Molecular cellbiology. 4th ed. New York: W.H. Freeman and Company2000, 496-531.5) Rao PN, Johanson RT, Mammalian cell fusion studieson the regulation of DNA synthesis and mitosis. Nature1970, 225: 159-164.6) Sunkara PS, Wright DA, Rao PN, Mitotic factors frommammalian cells induce germinal vesicle breakdown andchromosome condensation in amphibian oocytes. Proc NatlAcad Sci 1979, 76: 2799-2802.7) Maddaly Ravi, Deepa Parvathi. V, Govind Pai. M etal., Polyclonal antibody-mediated mitotic inhibition inChinese Hamster Ovary cells (CHO). J Can Res Ther 2006,3: 126-128.8) Hittelman WN, The technique of prematurechromosome condensation to study the leukemic process:review and speculations. Crit Rev Oncol Hematol 1986, 6:147-221.9) Rao PN, The phenomenon of premature chromosomecondensation. London; Academic Press Inc Ltd 1982, 2-40.10) Maddaly Ravi, Deepa Parvathi. V, Govind Pai. M etal., Evaluating homogeny of cellular mitotic cytosolic proteinantigens of Chinese Hamster Ovary (CHO), HeLa, Vero andhuman lymphocytes. Int J Can Res 2006, 4: 420-423.

Original Article


INTRODUCTION:Fluorescence in situ hybridization (FISH) refers to the

use of labeled nucleic acid sequence probes for thevisualization of specific DNA or RNA sequences on mitoticchromosome preparations or in interphase cells (Plate 1).Exciting advances in FISH are changing the nature ofcytogenetics, in both basic research and moleculardiagnostics. Cytogenetic analysis now extends beyond thesimple description of the chromosomal status of a genomeand allows the study of fundamental biological questions,such as the nature of inherited syndromes, the genomicchanges that are involved in tumorigenesis and the three-dimensional organization of the human genome. The highresolution that is achieved by these techniques, particularlyby microarray technologies such as array comparativegenomic hybridization, is blurring the traditional distinctionbetween cytogenetics and molecular biology. The currentreview focuses on the advances that FISH has undergone tosuit the requirements of present day diagnostics.

CHROMOSOME PAINTING AND ITS VERSATILITY IN MODERN DIAGNOSTICSHarpreet Kaur a, Teena Koshy a, Venkateswaran N a, Venkatachalam P a and Solomon F.D. Paul a

ABSTRACTThe last two decades have seen the advent of a novel andversatile technique, well known as Chromosome Paintingor Fluorescence In Situ Hybridization (FISH). During itsmaturation, various methodologies and modifications havebeen introduced to optimize the detection of DNA and RNA.The pervasiveness of this technique is largely because of itswide variety of applications and the relative ease ofimplementation and performance of in situ studies.


Dr. SOLOMON F.D. PAULDepartment of Human Genetics,Sri Ramachandra Medical College and Research Institute,Porur, Chennai – 600116email : [email protected] Department of Human Genetics

Chromosome painting allows precise visualization of uniquesequences, chromosomal sub-regions, or entire genome(DNA on metaphase chromosomes and interphase nuclei).It plays an increasingly important role in a variety of researchareas, including cytogenetics, prenatal diagnosis, tumorbiology, gene amplification and gene mapping. This reviewdescribes the applications of some FISH based techniquesin human disease diagnosis.Key words : Chromosome painting, nucleic acid hybridization

Probes and samples used for FISH:As mentioned earlier, FISH uses fluorescently labelled

probes for the visualisation of DNA sequences on metaphasespreads or interphase nuclei. Both numerical and structuralaberrations can be determined. Probes can be for the wholechromosome, centromere, or locus specific. FISH probes forthe entire genome are also often used. Interphase nucleican be obtained from a range of clinical specimens includingtouch preparations, fine needle aspirates, bone marrowsmears, and archival material (1).

Variations of FISH:Interphase FISHOne application of FISH involves the hybridization of probesto interphase cells. This is extremely beneficial when it isnot possible to prepare metaphase spreads as in the caseof primary tumors. In addition, interphase FISH can beperformed on paraffin-embedded, formalin-fixed tissuesections thereby allowing researchers to retrospectivelyanalyze samples and correlate chromosome aberrations withbiological and clinical endpoints. Interphase cytogeneticsalso allows one to precisely define the cell pool carryingchromosomal abnormalities, to identify whether aberrant

Plate - 1Step 1: Denaturation

Conversion of double stranded DNA into single strandedform

Denaturation of labeled probe DNA

Denaturation of target DNA(Interphase nuclei or metaphases on slide)

Step 2: HybridizationBinding of probe DNA to target DNA

Application of probe DNA to slide and overnight incubationat 37ºC

Step 3: Post hybridization washing and detection

Visualization of interphase nuclei / metaphases with boundprobe

Washing of unbound probe DNA, application of counterstainand visualization using fluorescence microscopy

Brief Report


cells exist in clonal patches or as isolated events and toobserve aberrations on a cell-to-cell basis rather than as apopulation (2).

Telomeric FISH (Q-FISH)Subtelomeric probes are a relatively new addition to

the arsenal of cytogenetic tests. This test is a collection of41 different FISH probes that are used to identifyrearrangements that cannot be seen by conventionalcytogenetic methods (3). The subtelomeric probes targetthe regions right behind the ends of the chromosomesthat enables to visualise if they are involved inrearrangements. Each of the probes is a different color sothat the specific chromosomal segment can be identified.This is a very expensive and labor-intensive process.However, it can be used when a geneticist suspects achromosomal abnormality and routine chromosomes arenormal (4) or when there is chromosomal material ofunknown origin.

RNA In Situ Hybridization (RISH)In many situations, transcription of genes at the cellular

level needs to be studied. Several groups have developedmethods for FISH of RNA. This is a potentially importantapplication of the FISH technique because it provides directvisual evidence of gene expression from a particularchromosome. House keeping genes, which are abundantlyexpressed, can be detected reliably. Further optimizationand amplification of the signal can even allow detection ofgenes expressed at baseline levels (5).

Primed In Situ Labelling (PRINS)The efficacy of FISH may be limited in specific

applications by low-resolution sensitivity. The primed insitu labelling (PRINS) method is an alternative to in situhybridization for chromosomal detection based on the useof chromosome-specific oligonucleotide primers. In thisprocedure, chromosomal identification is done by the insitu annealing of specific oligonucleotide primers, followedby primer extension by Taq DNA polymerase in the presenceof labelled nucleotides. It has been demonstrated that thePRINS technique is more specific and considerably fasterthan classical FISH for chromosomal identification (6)

Fiber FISH (Dynamic Molecular Combing)The term Fiber FISH refers to the common practice of

fluorescence in situ hybridization (FISH) conducted onpreparations of extended chromatin fibers. FISH on DNAfibers is useful in assessing the length of DNA probes, andto map probes relative to one another, as it can reveal eventheir degree of overlap. Thus, Fiber FISH has superiormapping resolution compared to interphase FISH. It canresolve DNA loci separated by a few kilobases and studyloci as large as two megabases in a single experiment (7; 8).

Comparative Genomic Hybridization (CGH)Comparative Genomic Hybridization serves as an

important global screening test for chromosomal aberrationspresent within a tumor genome. This technique requiresonly genomic tumor DNA and metaphase preps from a

normal donor, thus circumventing the preparation of high-quality tumor metaphase spreads. Tumor DNA extractedfrom archived, formalin-fixed paraffin-embedded tissue canalso be used. This allows identification of chromosomalaberrations and facilitates the correlation of cytogeneticfindings with histologic / histochemical information, clinicalcourse, and prognosis. Analysis of small subregions of ahistologically defined lesion is also possible (9). Onceregions of gain or loss have been identified, these regionscan be defined further using FISH or molecular genetictechniques (10). CGH coupled with microarray also knownas array CGH has proved to be very informative in manyclinical settings (11).

Combinatorial Binary Ratio Labeling (COBRA) FISHCombinatorial fluorescence in situ hybridization

(COBRA FISH) of the DNA of the 24 different humanchromosomes with 5 fluorophores in conjunction withspectral or filter-based microscopic imaging has greatlyadvanced molecular cytogenetic analysis of chromosomes.Use of 5 fluorophores allows the identification of up to 31different chromosomal targets on the basis of colorcombinations. (12) COBRA–FISH allows colordiscrimination of all of the p and q arms of each chromosomeand permits detection and elucidation of intra andinterchromosomal rearrangements (13)

Spectral karyotyping (SKY) FISHSpectral karyotyping (SKY) is a molecular cytogenetic

technique that allows differential visualization of all humanchromosomes in distinct colors with a single hybridizationand image exposure (14). After the chromosomes areclassified and aligned in a karyotype table, interpretationand comparison of all aberrations is summarized in thekaryogram.

Multiplex-FISH (M-FISH)The M-FISH technology has the ability to identify the twenty-four different human chromosomes in a metaphase spreadby the simultaneous hybridization of chromosome-specificDNA probes, each labeled with a different combination offluorescent dyes. M-FISH differs from SKY only in that it isa filter-based system where separate images are acquiredsequentially for each fluorochrome used. The individualfluorochrome files are then combined to generate the finalimage (15).

These techniques – M-FISH and SKY FISH, have combinedthe advantages of FISH with traditional chromosome bandingtechniques and spawned many variations resulting in diverseapplications. They permit the detection of interchromosomalstructural aberrations, such as translocations and insertionsresulting in balanced as well as unbalanced rearrangements.SKY and M-FISH have the potential to identify cryptictranslocations and clarify complex aberrations (marker andring chromosomes), which are typically unidentifiable byconventional banding techniques. In addition, otheraberrations such as double minutes can be better resolved,leading to the identification of critical oncogenes (16, 17).

Brief Report


Applications of FISHFISH is generally used either to complement classic

staining methods or a substitute for chromosomeidentification at metaphase or interphase. FISH has proveduseful in several clinical settings to determine prognosis.Discrete information is obtained for each cell, which is animportant advantage of the technique. In particular FISHdemonstrates the qualities listed below for various diagnosticand/or prognostic applications:

Sensitivity: FISH can detect cryptic chromosomal deletionsand rearrangements, not detectable by conventional means:submicroscopic microdeletions at the chromosomal level(18) and DNA level (19). FISH also helps in detection ofsingle gene disorders - Duchenne’s muscular dystrophy (20)and microduplications - mental retardation (21).

Specificity: By using a particular probe or probes,chromosomal material of unknown or uncertain origin canbe identified - marker chromosomes (22) and chromosomalvariants or polymorphisms (23).

Efficiency: FISH allows rapid screening of a large number ofmetaphases or interphases for a particular chromosome orother target sequence. Mosaicism (24) and chromosomalaneuploidies in prenatal samples (25) can be studied. FISHhas also proved invaluable in monitoring residual diseasestatus in patients with cancer (26).

Applicability: FISH allows interphase cells to be screenedfrom a wide variety of tissues not directly accessible withconventional cytogenetics. Some of the studied samples are:human lung carcinoma tissue (27), endometrial tissue (28)and uncultured chorionic villus samples (29). FISH may alsobe applied to buccal smear samples (30) where venous bloodis unavailable for cytogenetic analysis, or to blood smears,where an extremely rapid result is required (31). The FISHtechnique has also provided a great deal of information aboutchromosome behaviour at meiosis. FISH allows normal andabnormal chromosomes to be tracked through all stages ofmeiosis (32) Rapid, direct analysis of large numbers of thechromosomal complements of sperm, has been successfullyperformed using FISH by Chantot-Bastaraud et al (33).

Thus, FISH related applications allow information tobe mined irrespective of whether a single locus needs to bestudied or the entire genome needs to be scanned. Thefollowing section gives details on the impact of FISH oncertain disciplines.

CytogeneticsClassic cytogenetics has evolved from black and white

to technicolor images of chromosomes as a result of advancesin fluorescence in situ hybridization (FISH) techniques, andis now called molecular cytogenetics. Improvements in thequality and diversity of probes suitable for FISH, coupledwith advances in computerized image analysis, now permitthe genome or tissue of interest to be analyzed in detail ona glass slide. It is evident that the growing list of options forcytogenetic analysis has improved the understanding of

chromosomal changes in disease initiation, progression, andresponse to treatment. The contributions of classic andmolecular cytogenetics provided scientists and cliniciansalike with new avenues for investigation.

Small, submicroscopic, genomic deletions andduplications (1 kb to 10 Mb) constitute up to 15% of allmutations underlying human monogenic diseases. Novelgenomic technologies such as microarray-based comparativegenomic hybridization (array CGH) allow the mapping ofgenomic copy number alterations at this submicroscopiclevel, thereby directly linking disease phenotypes to genedosage alterations. At present, the entire human genomecan be scanned for deletions and duplications at over 30,000loci simultaneously by array CGH (approximately 100 kbresolution), thus entailing an attractive gene discoveryapproach for monogenic conditions, in particular those thatare associated with reproductive lethality. (34)

Fluorescence In Situ Hybridization (FISH) showed threesignals for chromosome X (green) and two signals forchromosome 18 (blue) confirming the karyotype results ofa structural anomaly - dicentric X.

Plate – 2Plate 2 shows the identification of a chromosome

anomaly by FISH in a case of primary amenorrhoea referredto the Department of Human Genetics for cytogeneticinvestigation.Fluorescence In Situ Hybridization (FISH) for chromosomes13 and 21 showed three signals for chromosome 21 (orange)and two signals for chromosome 13 (green) indicatingTrisomy 21 – Down’s Syndrome.

Plate – 3Plate 3 shows the confirmation of Down’s Syndrome –

Trisomy 21 by FISH, in a paediatric patient referred tothe Department of Human Genetics for cytogeneticinvestigation.

Brief Report


Prenatal DiagnosisPrenatal diagnosis employs a variety of techniques to

determine the health and condition of an unborn fetus.Numerical chromosome abnormalities are the major causeof inherited diseases with an incidence of 21% inspontaneous abortions. Of these, trisomies for sexchromosomes and chromosomes 13, 16, 18 and 21 accountfor 50% of chromosomally abnormal abortions. (35).

Prenatal diagnosis needs a rapid, accurate and overallgenome analysis. FISH is a powerful tool for detecting somegenetic diseases as well as microscopic or submicroscopicchromosome rearrangements in metaphases cells orinterphase nuclei involving chromosomes commonlyimplicated in aneuploidies - 13, 18, 21, X and Y (36).Interphase FISH is very useful in urgent high-risk cases. Theuse of FISH overcomes the difficulties of conventionalbanding on metaphase spreads. The ability to generateaccurate results in a few hours with FISH as compared tothe two weeks typically needed for standard karyotype analysishas been instrumental in relieving the anxiety of manywomen, or in allowing them, their families and theirphysicians to make difficult decisions more swiftly. It isusually employed as an adjunctive tool to conventionalcytogenetics (37).

Fluorescence In Situ Hybridization (FISH) forchromosomes 13 and 21 showed two signals forchromosome 21 (orange) and two signals for chromosome13 (green) indicating no numerical anomalies associatedwith chromosomes 13 and 21.

Plate – 4Plate 4 shows human amniocytes from amniotic fluid

probed by FISH in a case suspected as Down’s Syndrome(Trisomy 21), referred to the Department of Human Geneticsfor prenatal diagnosis.

Preimplantation GeneticsPreimplantation genetic diagnosis (PGD) identifies

genetic abnormalities in preimplantation embryos prior toembryo transfer. As mentioned earlier, the correlationbetween aneuploidy and declining implantation rates withmaternal age demands screening of chromosomeaneuploidies in human embryos by FISH using 13, 18, 21,X and Y probes should significantly reduce the risk of olderpatients undergoing in vitro fertilization (IVF) deliveringtrisomic offspring (35). PGD is being explored through polarbody biopsy, biopsy of the single cell from the eight-cell

embryo, and trophectoderm biopsy of the blastocyst (38).Interphase FISH using multi color, subtelomeric andcentromeric probes is used to test single cells for structuralor numerical chromosome abnormalities. It helps inidentifying embryos free of specific genetic abnormalities(39). Results with PGD indicate a significant decrease inspontaneous abortions, from 81% before PGD to 13% afterPGD (40).

Fluorescence In Situ Hybridization (FISH) forchromosomes 13 and 21 showed two signals forchromosome 21 (orange) and one signal for chromosome13 (green) indicating that the blastomere biospied from theembryo was abnormal.

Plate – 5Plate 5 shows FISH done on a blastomere obtained by

embryo biopsy prior to IVF performed for research purpose.

Cancer GeneticsChromosomal aberrations are found in cancer and tumor

cell lines. Some of them are already characterized andcorrelated with specific syndromes and some of them haveyet to be associated with a clinical outcome. Cytogeneticanalysis is now a routine part of the diagnosis andmanagement of a significant number of malignancies. Whilstconventional cytogenetics remains the most comprehensivemethod for assessing chromosome abnormalities, thetechnical difficulties associated with conventionalcytogenetics in most cancers has resulted in increased useof FISH to identify specific abnormalities that are useful ineither the diagnosis or management of these disorders.Aberrations such as aneuploidies, translocations, deletions,and gene amplifications are investigated in samples. This isaccomplished using probes for centromeres, wholechromosome probes, and/or probes for specific aberrantsequences (41).

In chronic lymphocytic leukemia (CLL), geneticanalyses by FISH and DNA sequencing have greatly improvedthe understanding of pathogenic events and prognosticmarkers (42). The combination of metaphase and interphaseanalyses and the investigation of specific structuralaberrations by FISH have definitely made tumor diagnosticsmuch rapid and accurate

Plate 6 shows the identification of the bcr-abl genefusion as identified by FISH and typical of Chronic MyeloidLeukemia (CML), in a patient referred to the Department ofHuman Genetics for cytogenetic investigation.

Brief Report


Fluorescence In Situ Hybridization (FISH) of interphase cellsshowed a fusion signal (yellow) confirming the presence ofPhiladelphia chromosome in a patient referred for chronicmyeloid leukemia (CML) with green / red signalsrepresenting normal chromosomes – 22 / 9.

Plate - 6

Radiation biodosimetryIn almost every instance involving accidental,

unexpected or suspected radiation exposure, biodosimetrycomes to the picture. Biodosimetry involves theidentification and scoring of certain biomarkers specificto and induced by radiation. To be useful, a biomarker forexposure and risk assessment should employ an end pointthat is highly quantitative, stable over time, and relevantto human risk (43). Biodosimetry is usually performed byenumerating the number of unstable chromosomeaberrations – Dicentric Chromosomes and Centric Ringsin peripheral blood lymphocytes of exposed individuals(44) Radiation induced unstable chromosomal exchangesare eliminated from the body within 1-3 years dependingon the exposure condition. As a result, there is aconsiderable uncertainty in this dosimetry for pastexposures (45, 46).

This is circumvented by scoring stable chromosomalexchanges such as translocations. Studies of the JapaneseA-bomb survivors and patients receiving radiotherapy haveshown translocations to persist in peripheral bloodlymphocytes many years after exposure and repeatedcytogenetic analyses have also indicated that thefrequencies of cells with translocations remain unchanged(47). Thus, they are potentially a better indicator ofcumulative dose.

The advent of Chromosome Painting (FISH) hasrevolutionized biodosimetry in simplifying the analysisof translocations. FISH assay not only makes theidentification of translocations very easy, but alsoincreases the sensitivity by its ability to score events,which conventional banding may fail to detect. Theusefulness of this technique is demonstrated by its abilityto resolve Complex chromosomal aberrations associatedwith high radiation doses, which are quite cumbersomeand time-consuming to deduce by routine conventionalcytogenetic techniques such as Banding by Trypsin andGiemsa (GTG Banding) (48).

Dual color FISH with whole chromosome and pan-centromere probes facilitates rapid detection oftranslocations. This approach allows analysis oftranslocations for assessment of genetic damage at long timesafter exposure or as a result of chronic exposure during along period of time. Multi-color FISH with locus specificprobes allows assessment of the frequency of cells carryingspecific aberrations known to be associated withtumorigenesis, analysis of the series of genetic changes thatoccur during tumor evolution and correlation betweengenotype and phenotype (49).

The metaphase shown below shows complexchromosomal aberrations involving chromosome 1 (green)and chromosome 3 (orange), an indication of high doses ofradiation exposure.

Plate – 7

Plate 7 shows some of the complex aberrations thathave been identified by FISH (Whole Chromosome Painting)in peripheral blood lymphocytes exposed to gammaradiation.

FISH - past, present and futureAlthough the basic principles of FISH have remained

unchanged, high sensitivity detection, simultaneous assayof multiple species, and automated data collection andanalysis have advanced the field significantly. Efficiency andsensitivity have been improved by combining methodologies.In the future, this technique is likely to have significantfurther impact on live-cell imaging and on medicaldiagnostics.

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2. Walter J, Joffe B, Bolzer A, et al. Towards many colorsin FISH on 3D-preserved interphase nuclei. Cytogenet.Genome. Res. 2006, 114(3-4): 367-78

3. Sismani C, Armour JAL, Flint J, et al. Screening forsubtelomeric chromosome abnormalities in children withidiopathic mental retardation using multiprobe telomericFISH and the new MAPH telomeric assay. Eur. J. Hum.Genet. 2001, 9: 527-32

Brief Report


4. Paoloni-Giacobino A, Dahoun S, Briault S, et al. Sixcases of cryptic subtelomeric translocations in four families:the use of subtelomeric FISH probes as a diagnostic tool.Genet. Couns. 2006, 17(1): 15-28

5. Dirks RW, van de Rijke FM, Fujishita S, et al.Methodologies for specific intron and exon RNA localizationin cultured cells by haptenized and fluorochromized probes.J. Cell. Sci. 1993, 104(4): 1187-97

6. Pellestor F, Girardet A, Andreo B, et al. The PRINStechnique: potential use for rapid Preimplantation embryochromosome screening. Mol. Hum. Reprod. 1996, 2(2):135-8

7. Raap AK, Florijn RJ, Blonden LAJ, et al. Fiber FISH asa DNA mapping tool. Methods. 1996, 9(1): 67-73

8. Weier HU and Chu LW. Quantitative DNA fibermapping in genome research and construction of physicalmaps. Methods. Mol. Biol. 2006, 338: 31-57

9. Tchinda J and Lee C. Detecting copy numbervariation in the human genome using comparativegenomic hybridization. Biotechniques. 2006, 41(4): 385,387, 389

10. Jarmuz M, Ballif BC, Kashork CD, et al. Comparativegenomic hybridization by microarray for the detection ofcytogenetic imbalance. Methods. Mol. Med. 2006, 128: 23-31

11. Rodriguez-Revenga L, Badenas C, Sanchez A, et al.Cryptic chromosomal rearrangement screening in 30 patientswith mental retardation and dysmorphic features. Clin.Genet. 2004, 65(1): 17-23

12. Raap AK and Tanke HJ. COmbined Binary RAtiofluorescence in situ hybridiziation (COBRA-FISH):development and applications. Cytogenet. Genome. Res.2006, 114(3-4): 222-6

13. Wiegant J, Bezrookove V, Rosenberg C, et al. BinaryRatio-labeling Fluorescence In Situ HybridizationDifferentially. Genome. Res. 2000, 10: 861-65

14. Schrock E, Zschieschang P, O’Brien P, et al. Spectralkaryotyping of human, mouse, rat and ape chromosomes -applications for genetic diagnostics and research. Cytogenet.Genome. Res. 2006, 114(3-4): 199-221

15. Cornforth MN, Greulich-Bode KM, Loucas BD, et al.Chromosomes are predominantly located randomly withrespect to each other in interphase human cells. J. Cell.Biol. 2002, 159(2): 237-44

16. Kearney L. Multiplex-FISH (M-FISH): technique,developments and applications. Cytogenet. Genome. Res.2006, 114(3-4): 189-98

17. Dorritie K, Montagna C, Difilippantonio MJ, et al.Advanced Molecular Cytogenetics in Mouse and Human(FISH, SKY and CGH). Expert. Rev. Mol. Diagn. 2004, 4(5):663-76

18. Yu S, Baker E, Hinton L, et al. Frequency of truly crypticsubtelomere abnormalities - a study of 534 patients andliterature review. Clin. Genet. 2005, 68(5): 436-41

19. Engelen JJM, Albrechts JCM, Loots WJG, et al.Applications of microfish to delineate deletions. Cytogenet.Cell. Genet. 1996, 75: 167–71

20. Stuppia L, La Sala D, Cinti C. Combined fluorescencein situ hybridization and PRINS for the analysis of theDystrophin gene. Methods. Mol. Biol. 2006, 334: 115-22

21. Mao R, Pevsner J. The use of genomic microarrays tostudy chromosomal abnormalities in mental retardation.Ment. Retard. Dev. Disabil. Res. Rev. 2005, 11(4): 279-85

22. Kolialexi A, Kitsiou S, Fryssira H, et al. Identificationof autosomal supernumerary chromosome markers (SMCs)by fluorescent in situ hybridization (FISH). In. Vivo. 2006,20(4): 473-8

23. Ungaro P, Christian SL, Fantes JA, et al. Molecularcharacterisation of four cases of intrachromosomaltriplication of chromosome 15q11-q14. J. Med. Genet.2001, 38(1): 26-34

24. Siffroi JP, Dupuy O, Joye N, et al. Usefulness offluorescence in situ hybridization for the diagnosis of Turnermosaic fetuses with small ring X chromosomes. Fetal. Diagn.Ther. 2000, 15(4): 229-33

25. Amiel A, Goldzak G, Gaber E, et al. Molecularcytogenetic characteristics of Down syndrome newborns. J.Hum. Genet. 2006, 51(6): 541-7

26. Schoch C, Schnittger S, Bursch S, et al. Comparison ofchromosome banding analysis, interphase andhypermetaphase FISH, qualitative and quantitative PCR fordiagnosis and for follow-up in chronic myeloid leukemia: astudy on 350 cases. Leukemia. 2002, 16(1): 53-9

27. Halling KC, Rickman OB, Kipp BR, et al. A comparisonof cytology and fluorescence in situ hybridization for thedetection of lung cancer in bronchoscopic specimens. Chest.2006, 130(3): 694-701

28. O’Toole SA, Dunn E, Sheppard BL, et al. Genome-wide analysis of deoxyribonucleic acid in endometrial cancerusing comparative genomic hybridization microarrays. Int.J. Gynecol. Cancer. 2006, 6(2): 834-42

29. Goumy C, Bonnet-Dupeyron MN, Cherasse Y, et al.Chorionic villus sampling (CVS) and fluorescence in situhybridization (FISH) for a rapid first-trimester prenataldiagnosis. Prenat. Diagn. 2004, 24(4): 249-56

30. Harris C, Wilkerson C, Clark K, et al. Potential use ofbuccal smears for rapid diagnosis of autosomal trisomy orchromosomal sex in newborn infants using DNA probes.Am. J. Med. Genet. 1994, 53(4): 355-8

31. McKeown CME, Waters JJ, Stacey M, et al. Rapidinterphase FISH diagnosis of trisomy 18 on blood smears.Lancet 1992, 340(8817): 495

Brief Report


32. Oliver-Bonet M, Benet J, Martin RH. Studying meiosis:a review of FISH and M-FISH techniques used in the analysisof meiotic processes in humans. Cytogenet. Genome. Res.2006, 114(3-4): 312-8

33. Chantot-Bastaraud S, Ravel C, Berthaut I, et al. Sperm-FISH analysis in a pericentric chromosome 1 inversion,46,XY,inv(1)(p22q42), associated with infertility. Mol.Hum. Reprod. 2006 [Epub ahead of print]

34. Vissers LE, Veltman JA, van Kessel AG, et al.Identification of disease genes by whole genome CGHarrays. Hum. Mol. Genet. 2005, 14(2): 215-23

35. Munne S. Preimplantation genetic diagnosis ofnumerical and structural chromosome abnormalities. Reprod.Biomed. Online. 2002, 4(2): 183-96

36. Lespinasse J, Nadeau G. Molecular chromosomicgenetics in prenatal and perinatal diagnosis of chromosomalanomalies and genetic diseases. Presse. Med. 2005, 34(17):1257-63

37. Jobanputra V, Roy K K and Kucheria K. Prenataldetection of aneuploidies using fluorescence in situhybridization: A preliminary experience in an Indian setup. J. Biosci. 2002, 27: 155–163

38. Simpson JL. Preimplantation genetics and recovery offetal cells from maternal blood. Curr. Opin. Obstet.Gynecol. 1992, 4(2): 295-301

39. Kearns WG, Pen R, Graham J, et al. Preimplantationgenetic diagnosis and screening. Semin. Reprod. Med. 200523(4): 336-47

40. Munne S. Analysis of chromosome segregation duringpreimplantation genetic diagnosis in both male and femaletranslocation heterozygotes. Cytogenet. Genome. Res. 2005,111(3-4): 305-9

41. Schwanitz G, Raff R. Application of specific cytologic,cytogenetic and molecular-cytogenetic techniques for thecharacterization of solid tumors. Rocz. Akad. Med. Bialymst.2005, 50: 91-6

42. Stilgenbauer S, Dohner H. Genotypic prognosticmarkers. Curr. Top. Microbiol. Immunol. 2005, 294:147-64

43. Lucas JN. Chromosome Translocations: A Biomarkerfor Retrospective Biodosimetry Environ. Health. Perspect.1997, 105(6): 1433-6

44. Brooks AL. Biomarkers of exposure, sensitivity anddisease. Int. J. Radiat. Biol. 1999, 75(12): 1481-1503

45. IAEA, Biological Dosimetry: Chromosome aberrationanalysis for dose assessment. 1986, STI/PUB/10/260(Vienna: IAEA)

46. Sreedevi B, Rao BS and Bhatt B. Radiation inducedchromosome aberration yields following an accidental non-uniform exposure. Radiat. Prot. Dosim. 1993, 50: 45-9

47. Amundson SA, Bittner M, Meltzer P, et al. Biologicalindicators for the identification of ionizing radiation exposurein humans. Expert. Rev. Mol. Diagn. 2001, 1(2): 211-9

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Brief Report


INTRODUCTIONBorn in secrecy and viewed with skeptism,

Laparoscopic cholecystectomy (LC) stormed into thesurgical arena, and in an unimaginable span of time hasbecome the gold standard, for the treatment of Gall stonedisease. The benefits of LC were so obvious that, till nowthere are no Randomized Controlled Trial comparing LCand Open Cholecystectomy (OC). In a lighter vein, thehighest level of evidence (class A) is lacking, to supportthe advantages of LC. The incidence of bile duct injury inLC is 0.4% - 1.3 %(1,2,3,4,5,6) compared to 0.2%-0.3%(6,7) for OC. Despite the inherent difficulty incollecting data on laparoscopic bile duct injuries, theseinjuries occur twice more frequently in Laparoscopiccholecystectomy. This statistical data may look innocuous,but nevertheless there are major differences between thebile duct injuries associated with LC and OC.

Are Post laparoscopic Bile duct injuries anyway different1. The so-called “classical Laparoscopic injury” (8) ischaracterized by significant segmental loss of bile duct,and often the proximal level of injury may reach thebiliary confluence. This is because, the surgeon dividesthe CBD mistaking it for the Cystic duct, and further inthe process of removing the GB, he divides the CHDproximally and finally ending up in a significant loss oflength. Basically the cranial traction exerted on the GBfundus to lift the Liver makes the Cystic duct to alignwith the CBD and hence the mistake. This complex kindof injury is unique for Laparoscopy and unlikely tohappen in OC.2. Many a time bile duct injuries are associated with RightHepatic Artery injury (RHAI) because of its close proximity

LAPAROSCOPIC BILE DUCT INJURIES-CONTROVERSIES AND CONSENSUSS. Sankar a, M. Subramanian a

ABSTRACTAmong the various advancements witnessed in the field

of surgery towards the end of the last millennium, none hasbeen so dramatic as Laparoscopy Cholecystectomy.Laparoscopic cholecystectomy is the prototype amongst theminimally invasive surgeries; however this unprecedentedrapid advancement was plagued by the increased incidenceof bile duct injuries. Frequency of bile duct injuries isalmost double in Laparoscopy. The mechanism of injury isdifferent. Laparoscopic anatomy is different. Though therecognition of injury is relatively early, the magnitude ofinjury can be devastating. The implications of post-

laparoscopic cholecystectomy biliary injuries are serious interms of the physical and fiscal inconvenience for the patientsand the legal problems for the surgeon. For obvious reasons,accrual of data on this entity is extremely difficult. Despitethe completion of learning curve, bile duct injuries continueto occur. This article reviews the controversies and consensuson the subject of post-laparoscopic cholecystectomy biliaryinjuries.

Key words:

Laparoscopic, Cholecystectomy, Bile duct diseases

to the CHD, which is invariably damaged in the classicalLC bile duct injury. Normally it is thought that RHAI leadsto increased incidence of complications like liver abscess,necrosis, bile leaks, unsuccessful repairs etc. In aretrospective study of 261 LC bile duct injuries, Stewartand Way analyzed the mechanism and consequences ofRHAI (9). The incidence of RHAI was an amazing 32%,but ironically this was not associated with increasedmortality, and did not alter the success of repair in the handsof experienced biliary surgeons (9). Belghiti’s group has alsoreported the incidence of RHAI to be 36%(40). Most of thetimes the RHAI goes unrecognized. If the injury is recognizedon the table, whether one should try to reconstruct is amatter of debate. But in the presence of pre-existing Liverdysfunction, Right Hepatic artery injury may have seriousconsequences.

3. Many LC biliary injuries are due to surgeons’misperception of the anatomy rather than an inadequacy ofskill or judgment (10). Without realizing this optical illusion,the surgeon subconsciously feels that, what he was doingwas correct. In cognitive psychology this is technically calledthe “Heuristic process”. This means, biliary injuries willcontinue to occur, despite the completion of the often-repeated cliché, “learning curve”. But this should not bemisinterpreted as a justification for the errors, that carriestremendous physical, emotional, legal and financialimplications. There have been suggestions to inculcate“methods of error reduction” in the laparoscopic training ofa surgeon, something similar to that followed in sectorslike aviation, nuclear technology, as errors in these fieldscan have serious consequences.

Laparoscopic Anatomy – is it different?

• The commonly described Calot’s triangle is actuallydifferent from what it was originally described. In theoriginal Calot’s triangle, Cystic artery forms the boundaryand not the content. The Hepato-Cystic triangle (alsocalled as Mooseman’s area or triangle of Buddie), is theone where Cystic artery forms the content and Liver formsa boundary.


Dr. S. SANKARAssistant Professor of Surgical GastroenterologySri Ramachandra Medical College and Research InstitutePorur, Chennai - 600 116.E-mail: [email protected] Department of Surgical Gastroenterology

Review Article


• The cranial traction applied on the fundus of GB toretract the Liver, actually narrows the Calot’s Triangle andhence an outward traction on the Harman’s pouch is neededand causes the Cystic duct to align with the CBD

• The “reverse” or “posterior” dissection of the Calot’striangle gives a different view of the anatomy

• “Rouvier Sulcus” can be demonstrated in a majority ofpatients, this sulcus separates the right lobe and caudateprocess and corresponds to the porta hepatis harboring righthepatic pedicle. Hence the dissection should start anteriorto the sulcus. Thus it provides an extra biliary referencepoint, which does not get altered due to the pathology ofGall bladder (41).

• In LC, the junction of cystic duct and Hartman’s pouchis demonstrated. Whereas in OC the junction of Cystic ductand CBD is demonstrated.

Factors predisposing for biliary injuries include

A. Difficult anatomy

i. Anomalies of cystic duct (low insertion, high insertion,short duct, parallel duct)

ii. Right sectoral duct anomalies (in 20% of the cases oneof the sectoral duct join the CHD)

iii. Intrahepatic GB

iv. Arterial anomalies (accessory Right hepatic artery,arterial humps)

B. Difficult pathology

i. Acute cholecystitis

ii. Scleroatrophic GB (contracted GB)

iii. Mirrizi syndrome

iv. Chronic cholecystitis

v. Frozen calots’s triangle

C. Difficult in terms of techniquei. Casual attitude towards a “simple Gall bladder”

ii. Improper placement of trocars

iii. Bulky and hanging falciform ligament

iv. Bulky Quadrate lobe

v. Undue haste exercised in clamping or clipping in theevent of bleeding

vi. Injudicious use of electro cautery in Calot’s triangle

vii. Too much skeletonization of the Bile duct leading toischemic strictures

viii. Surgeons’ experience- the “learning curve effect”,which implies that the bile duct injuries andexperience of the surgeon are inversely proportional.There is considerable evidence to support this view(2, 5, 11, 12, 13). However there are also reports ofmajor injuries, inflicted by surgeons of considerableexperience (14)

Classification

Bismuth classification (15) is shown in figure 1.This is used for strictures but the main drawback is, it

does not include all possible biliary injuries duringLaparoscopic Cholecystectomy.

Fig. 1

Type I Stricture > 2cm from the confluence. More than2 cm of CHD is available.

Type II Stricture < 2 cm from the confluence. Less than2 cm of CHD is present.

Type III No CHD, but the confluence is patent and theright and left systems are communicating.Sikora et al have proposed a sub classificationof type III strictures. III A, where both the floorand roof of the confluence are healthy. III B,where the roof of the confluence is healthy,but the floor is scarred (24). Their contentionis, this sub classification has therapeutic andprognostic significance. Type III B stricturesare difficult to treat and resembles that of typeIV strictures.

Type IV Confluence is stricturous. The two systems areisolated.

Type V Stricture at the junction of the aberrant sectoralduct.

Review Article


Strasburg classification (6) is represented in figure 2.This classification includes all possible types of

injuries, both leaks and strictures.

Fig. 2

Type A leak from minor ducts like Cystic duct or duct ofLushka

Type B Ligation of aberrant right sectoral ductType C Division of aberrant right sectoral ductType D Lateral injury the major extra hepatic ductType E Classical LC bile duct injury E1, E2, E3, E4, E5

(as per Bismuth classification)

Stewart & Way classification (16) (figure 3)This is more practical based on the different operative

scenario, where injuries occur.

Fig. 3

Class I CBD mistaken for Cystic duct, but recognizedby the surgeon

Class II Lateral damage to CHD due to clips or cauteryClass III CBD mistaken for cystic duct. CBD, CHD

resectedClass IV RHD mistaken for CD, RHA mistaken for CA,

RHD, RHA transected. Lateral damage to RHDdue to clips or cautery

Clinical presentationDepending on the temporal relation with the injury,

three situations are possible, namely injuries recognized onthe table, immediate postoperative period and delayedpresentations.

1. At surgery:The surgeon recognizes this by finding welling of bile

in the operative field (excluding the bile spillage from theGB). Ideally at this point surgeon should convert and mayrequest for expert help. In some cases bile duct injuriesmay become evident after an intra-operative cholangiography.Non-visualization of proximal Ductal system indicates majorinjury. Uncontrollable bleeding also mandates conversioninstead of blindly clipping and cauterizing. If any one ofthe major ducts is clipped and if it is recognized immediately,the clip should be removed and usually nothing more isneeded.

The role of intra-operative cholangiogram, whetherroutine or selective has been a matter of debate. Intra-operative cholangiography may only help to identify injuriesand correct it, but it is unlikely to prevent occurrence ofinjuries (36). There are reports of low yield of intra-operativecholangiography and its routine use is discouraged (37,38,39). Selective use of intra-operative cholangiography ispreferred. When the anatomy is distorted, intra-operativecholangiography is indicated and it is recommended toconvert to OC, since Laparotomy is less dangerous thanundetected biliary injury.

2. Injuries detected in the immediate post operative periodBile duct injuries sustained in Laparoscopic

cholecystectomy are likely to be recognized relatively earlierthan its counterpart, namely open cholecystectomy (8, 17),which probably reflects the differences in the pattern ofinjuries and the surgeon’s awareness of the “potential oflaparoscopy” in inflicting injuries (6)

On the first post-operative day, the patient should be“one hundred percent fine” in an otherwise smooth LC,and anything short of this recovery should alert the surgeon.The clinical features depend on various factors. Whether itis a bile leak or an occluded duct, occlusion may be partialor complete. Leaks may be “minor or major” and “containedor uncontained”. Strasburg A, C, D and E are likely to presentwith leaks. Clinical features also depend on whether a drainis placed or not and whether the drain is draining or not.Most of the times drains help in detecting bile leaks. But,“a drain is useful only if it drains” and should not be analternative to clinical monitoring. Drains can get blockedand can provide a false sense of security. Significant bile inthe drain necessitates the surgeon to order for ultrasonogram.The use of drains in laparoscopic cholecystectomy shouldbe selective. Sometimes patients are discharged early andget readmitted with features of sepsis.

Tachycardia, fever and general weakness may be thesubtle signs of bile leak. In grossly obese individuals often

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the signs of peritonitis are absent and one has to rely onindirect evidences (in these patients, a sense of “impendingdoom” may be an indicator of a serious problem). Bile leaksmay range from being trivial to massive. Large subhepaticcollections may compress the vena cava; reduce the preloadto the heart and a fall in the systolic pressure (WaltmanWalter syndrome). Undetected or neglected massive bileleaks may be fatal especially when they are uncontainedand if the patient is allowed to slip into sepsis and itssequelae.

When the extra hepatic bile duct is occluded, patientpresent with varying degree of icterus and rising enzymesand vague abdominal pain. Usually it takes more than twoweeks for the IHBR dilatation to occur. Isolated sectoralduct occlusion may not manifest with jaundice, but onlyenzymes are elevated.

Late presentationOccurs typically in biliary stricture patients. Recurrent

fever with mild icterus characterizes this group of patients.Recurrent cholangitis leads to general ill health and malaise.Often the bilirubin is elevated to moderate levels. Onceagain clinical features depend on the magnitude of occlusionand the location of stricture. Strictures of the right sectoralducts may lead to recurrent infections, abscesses or atrophyof liver sector. Long standing biliary obstructions maypredispose to secondary biliary cirrhosis and portalhypertension. Presence of portal hypertension may also bedue to associated portal venous injury. Total occlusion ofthe duct may cause deep jaundice. Sometimes an elevatedalkaline phosphatase may be the only abnormality. Evenasymptomatic or minimally symptomatic patients may needtherapeutic intervention, as secondary biliary cirrhosis islikely to occur.

Pathological consequences of biliary stricture

1. Fibrosis:High local concentration of bile salt at the level of

canalicular membrane sets a cascade of molecular andcellular changes, leading to fibrosis and scarring of bile ductsand ductules, which may sustain the cholestasis. Howeverthe fibrosis is not equivalent to cirrhosis as the liverarchitecture is maintained. Early drainage of the liver mayproduce reversal of changes and in this context a concept of“latent portal hypertension” has been proposed by some(18). In one study, duration of biliary obstruction and thetrend of ALT levels were independent predictors of Hepaticfibrosis (32)

2. AtrophyThe liver mass is regulated by mechanisms that are

poorly defined, but biliary drainage and portal venous flowhas a bearing on it. Sectoral or segmental ductal obstructionmay lead to atrophy of the corresponding segments. This isassociated with contralateral lobe hypertrophy. Atrophy-hypertrophy complex is clinically significant as it hasimplications in diagnosis and surgical treatment. Atrophy-

hypertrophy complex leads to rotation of the hepatoduodenalligament leading to technical difficulties for the surgeon asthe portal vein may be encountered more anteriorly.

3. Portal hypertensionThe presence of portal hypertension in biliary stricture

may be due to one of the following three reasons.

1. Secondary biliary cirrhosis

2. Portal vein injury

3. Pre-existing liver disease.

It takes nearly 2 years for the secondary biliary cirrhosisto contribute to portal hypertension. In alcoholics this periodmay be shortened. The concept of “Reversible” or “Latentportal hypertension” is interesting (18). Whatever may bethe etiology of portal hypertension, it carries a poorprognosis, and for the surgeon, enough of technicaldifficulties. In the presence of portal hypertension someprefer a two-stage approach i.e.: a porto systemic shuntfollowed by hepaticojejunostomy or a single stage withvenovenous bypass (19). Some prefer single stage procedurewith acceptable morbidity, ie: Hepaticojejunostomy withouta preliminary shunt surgery (20)

ManagementManagement depends on the scenario of injury. Three

situations are possible.

1. On the table

2. Immediate postop period

3. Late (Biliary stricture)

Intra operatively detected injuriesUsually bile is found welling in the operative field or

the anatomy is distorted and uncomfortable. An operativecholangiogram is indicated at this point to find out thecause of leak. Non-filling of proximal ducts oncholangiogram may also indicate CBD injury. Mostly thissituation warrants conversion and the need for help. It isbest to accept that any potential morbidity from a laparotomyis minor compared to a bile duct injury.

1. CBD transected, but no segmental loss.If the CBD has been transected and there is no

segmental loss, the best option is to do aCholedochojejunostomy. But fashioning a bilio entericanastomosis in an undilated duct may be technicallydemanding. The other option is to do a primary repair witha T tube brought out separately. Though primary repair ofCBD is associated with high rate of stricture formation (50%of cases), these strictures are the ones, which can best bemanaged with endoscopic methods. The high rate of strictureafter primary repair is due to the axial blood supply, 60%coming from distal side.

2. CBD transected with segmental loss.If CBD transected with segmental loss then it warrants

a hepaticojejunostomy. If the surgeon is not confident or

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no expertise is available, a good drainage is accomplishedand patient is referred to a higher center where services of aHepatobiliary surgeon is available.

3. Lateral injuries.Lateral injuries of the CBD once again need biliary

enteric anastomosis, as suturing over a “T-tube” maycompromise the lumen and end up in a stricture. Inadvertentapplication of clips on the bile duct if recognized,immediate removal will suffice.

Immediate postoperative periodReports of successful early repair of biliary injuries

continue to appear (21). But the key issue is “a stable patientand absence of sepsis”. The first priority in biliary injuriesdetected in the immediate postoperative period is to stabilizethe patient hemodynamically. An USG abdomen is done toknow whether the leak is contained or not. A CT is betterthan USG, because ultrasound is less sensitive in theevaluation of post operative fluid collections. If the leak isnot contained, it has to be contained by proper drainage,either by a Radiological procedure or may be aRelaparoscopy. Once a leak is contained and the patient ishemodynamically stable, the next question to be answeredis whether the biliary continuity is preserved or not.Non-invasive methods of knowing this is by MRCP orIsotope scan. MRCP and contrast (Mangafodipir) enhancedMRCP are increasingly being used in the evaluation of biliaryinjuries and are very reliable (22,23). Isotope scan suffersfrom the drawback of not being an anatomical investigation,but when the duodenum shows activity, it is indirectlyinferred that biliary continuity is maintained. Havingestablished the biliary continuity, the best option is to doan ERCP. If the leak is due to cystic duct blow out a stent ora nasobiliary drain with or without a sphincterotomy willsettle the issue. With this modality the leak settles veryquickly. Since majority of the cystic duct leaks tend to heal,some surgeons tend to treat conservatively without an ERCP,as long as the patient is stable without signs of sepsis. Butthe inherent problem with this approach is that the surgeonpresumes that bile leak is from the cystic duct and not frommajor Ductal injury. Conservative treatment without an ERCPmay also prolong the hospital stay.

When the biliary continuity is lost (classical LC bileduct injury), and the surgeon is experienced, earlyhepaticojejunostomy is accepted. “How early is early” maydepend on the surgeon’s philosophy and experience, apartfrom the logistics. In delaying, Hepaticojejunostomy maybe postponed to a later date after containing the leak, as thefirst repair should be the best repair, and with every furtherattempt, surgery becomes less successful and more difficult.

Late presentationThis is essentially the typical biliary stricture. A good

cholangiogram is an essential mandatory preop investigation.MRCP is an ideal modality and has almost replaced PTC(31) as it gives all the information that the surgeon wants,

besides being non invasive. Of course the gold standard isPTC, which provides a good quality when compared toMRCP. If the patient is having cholangitis that is notresponding, one may have to embark on a PTBD. RoutinePTBD is not needed. When one decides to do a PTC, it istimed just before the surgery for fear of cholangitis.

The basic principle in the treatment of biliary strictureis to relieve the symptoms and prevent the developmentof secondary biliary cirrhosis. Surgery has been the standardtreatment and the gold standard against which othertreatments are compared. Any treatment modality shouldbe durable and long lasting with less morbidity and nomortality. Surgery always carries morbidity and the remotepossibility of mortality. The results of the surgery arevariable. In the hands of experienced biliary surgeonsresults have been excellent. Long-term success rate of 80-90% has been reported from high volume centers (33, 34,35). Biliary sepsis and portal hypertension are two factorshindering surgical success. The recurrence of stricture isreported in 10% of the patients. Treatment of choice forrecurrent strictures after surgery is balloon dilatation. Somecases of recurrent stricture may warrant redoHepaticohejunostomy.

A good Hepaticojejunostomy is the treatment of choicefor biliary stricture. The classical Hepp Couinaud approachwherein a wide opening is made in the dilated CHD andextending onto the left duct and a side-to-side anastomosisis fashioned to a Roux Loop of Jejunum. In a nutshell thefollowing are the principles of surgery.

• Careful meticulous dissection

• Duodenum, Colon and Stomach will be adherent tothe GB fossa, which are taken down.

• Identification of dilated bile duct

• Lowering of hilar plate

• Bile duct opened and the incision is extended on tothe left duct for a wide anastomosis

• Roux loop of jejunum is fashioned

• Single layer interrupted, mucosa to mucosa anastomosiswith Vicryl or PDS

• Access loop in selected cases

• Appropriate Biliary stent and drains

• Long follow up

Surgery vs. EndoscopyTraditionally management of biliary stricture has been

a Surgeon’s domain. An Endoscopist has a definite role toplay in the rescue of the surgeon in the immediatepostoperative period. With the advancements of endoscopictechnique and accessories, endoscopic management isemerging as an alternative modality to surgery in themanagement of established biliary strictures. There are fewstudies directly comparing the results of surgery andendoscopic treatment. The available data is from retrospective

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series. The studies showed endoscopy to be comparable withsurgery, except for the fact that endoscopy could producesuch an impressive results only by “repeated stenting” and“multiple stents”(25,26,27). Endoscopy has a high failurerate in hilar strictures. It is interesting to note that, reportsof self-expanding covered metal stent have appeared in thescene (28). These stents can be removed after it has servedthe purpose. The concept seems to be appealing, but wehave to wait for the long-term results. Bioabsorbable stentsmay have an important role to play in the future (29)

CONCLUSIONS:Biliary injury is the Achilles’ heel of Laparoscopic

Cholecystectomy. William Halsted, the great Surgeonsustained biliary injury during Cholecystectomy andsuccumbed to it after resurgery. Anthony Eden one of theyoungest prime minister of Great Britain had his politicalcareer curtailed, as he became a biliary cripple followingcholecystectomy and subsequent repeated operations.However these were in the prelaparoscopic era. Despite allthe precautions and learning curves, bile duct injuries willcontinue to occur, and be the nemesis for the LaparoscopicSurgeon. Errors happen at so many levels, but it is up to theSurgeon who is the last level in the Swiss cheese model,not to allow it to pass through him. Management of suchcomplex problems should integrate the services of Hepato-biliary Surgeon, Interventional Endoscopist, Interventionalradiologist and the Intensivist, to optimize the results.

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3. Fletcher D R, Hobbs M S T, Tan P, Valinsky L J, HockeyR L, Pickora T J, Knuiman M W, Sheiner H J, Edis AComplications of Cholecystectomy: risks of Laparoscopicapproach and protective effects of operative cholangiography.Annals of Surgery 1999:229:449-457

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6. Strasburg S M, Hertl M, Soper N J An analysis of theproblem of biliary injury during Laparoscopiccholecystectomy. Journal of American College of Surgeons1995:180:101-125

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8. Davidoff A M, Pappas T N, Murray E A, Hilleren d J,Johnson R D, baker M E, Newman G E, cotton P B, MeyersWC. Mechanisms of major biliary injury during laparoscopiccholecystectomy. Annals of Surgery.1992: 215:196-208

9. Stewart L, Robinson TN, Lee CM, Liu K, Whang K,Way LW. Right hepatic artery injury associated withlaparoscopic bile duct injury: incidence, mechanism, andconsequences. J Gastrointestinal Surg 2004 Jul-Aug;8(5):523-30

10. Way LW, Stewart L, Gantert W, Liu K, Lee CM, WhangK, Hunter JG Causes and prevention of laparoscopic bileduct injuries: analysis of 252 cases from a human factorsand cognitive psychology perspective. Ann Surg. 2003 Apr;237(4): 460-

11. Gigot J, Etienne J, Aerts R, Wibin E, Dallemagna B,Deweer F, Fortunati D et al The dramatic reality of biliarytract injury during laparoscopic cholecystectomy. Ananonymous multicenter Belgian survey of 65 patients.Surgical Endoscopy 1997:11:1171-1178

12. Woods M S, Traverso L W, Korzareck R A, Tso J I,Rossi R L, Gough D, Donahue J H Characteristics of biliarytract complications during laparoscopic cholecystectomymulti-institutional study. American Journal of Surgery 1994;167; 27-34

13. Russel J C, Walsh S J, Mattie A S, Lynch j T bile ductinjuries, 1989-1993: a statewide experience-Connecticutlaparoscopic cholecystectomy registry. Archives of Surgery1996:131; 382-388.

14. Carroll B J, Friedman R L, Liberman M a, Phillips E HRoutine Cholangiography reduces sequelae of Commonbile duct injuries. Surgical Endoscopy1996: 10:1194-1197

15. Bismuth H Postoperative strictures of the bile duct. In:Blumgart LH(ed) The Biliary tract. Clinical surgeryinternational. Churchill Livingstone, Edinburgh. 1982:209-218

16. Lygia Stewart et al. Surgical management ofHepatobiliary and Pancreatic disorders; Poston &Blumgart(ed). Martin Dunitz, London and New York.2003:316-319

17. Lillimoe K D, Martin S A, Cameron J L, Yeo C J,Talamini M A, Kaushal S, Coleman J, Pitt H A et al.Major bile duct injuries during laparoscopiccholecystectomy; follow up after combined surgical andradiological management. Annals of Surgery: 1997:225:459-471

18. Ibrarullah M, Sikora S S, Agarwal D K, Kapoor V K,Kaushik S P. Latent portal hypertension in benign biliaryobstruction. HPB Surgery; 1996:9:149-152

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19. Christophi C, Dudley F, McInnes I E. Surgical treatmentof biliary strictures with portal hypertension usingvenovenous bypass. Aust N Z J Surgery; 1991:61(4):316-318

20. Perakath B, Sitaram V, Mathew G, Kandhuri P.Postcholecystectomy benign biliary strictures with portalhypertension: Is a Porto systemic shunt beforehepaticojejunostomy necessary. Ann R Col Sur Eng.2003:85(5): 317-20

21. Thomson B N J, Parks R W, Madhavan K K, WigmoreS J, Garden O J. Early specialist repair of biliary injury; BJS:2006:93:216-220

22. Tahir R K, Javier C V, Berta M M, Raul C, Joe U L.Using MR Cholangiopancreatography to evaluate iatrogenicbile duct injury. AJR; 2001:177:1347-1352

23. Aduna M, Larena J A, Martin D et al. AbdominalImaging; 2005:30:480-487

24. Sadiq S Sikora, Gadiyaram Srikanth, Anuj Sarkari, AshokKumar, Rajan saxena, Vinay K Kapoor. Hilar benign biliarystricture; need for sub classification. ANZ journal of surgery;2003:73:484

25. Davids P, Tanka A, Rauws E A, Van Gulik T M, et al.Benign biliary strictures: Surgery or Endoscopy. Annals ofsurgery: 1993:217:237-43

26. Costamagna G, Pandolfi M, Mutignani M, Spada C,Perri V. Long-term results of Endoscopic management ofpostoperative bile duct strictures with increasing number ofstents. Gastrointestinal Endoscopy; 2001; 54:162-8

27. Draganov P, Hoffman B, Marsh W, Cotton P,Cunningham J. Long-term outcome to patients with benignbiliary strictures treated endoscopically with multiple stents.Gastrointestinal Endoscopy 2002; 55:680-6

28. Kahaleh M, Tokar J, Le T, yeaton P. Removal of selfexpandable metallic wall stents. Gastrintest Endosc 2004;60:640-4

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30. Chaudhary A, Manisegran M, Chandra A, Agarwal A,Sachdev A K. Do bile duct injuries following Laparoscopiccholecystectomy differ from those following opencholecystectomy. J Laproendosc Adv Surg Tech.2001;11:187-191

31. Chaudhary A, Negi S S, Puri S K, Narang P.Comparison of Ptc and MRCP in the evaluation of postcholecystectomy bile duct injuries. Br J Surg 2002;89:433-6

32. Negi S S, Sakhuja P, Malhotra V, Chaudhary A. Factorsresponsible for hepatic fibrosis in patients withpostcholecystectomy bile duct injuries Arch Surg 2004;139:299-303

33. Bismuth H, franco D, Corlette M B, hepp J. Long-termresult of Roux en Y hepaticojejunostomy. Surg gynaecologyObstet 1978; 146:161-7

34. Chapman W C, Havely A, Blumgart L H, Benjamin IS. Postcholecystectomy bile duct strictures: managementand outcome in patients. Arch Surg 1995; 130:597-604

35. Murr M M, Gigot J F, Nagorney D M, harmsen W S,Ilstrup D M, farne M B. Long-term result of biliaryreconstruction after laparoscopic bile duct injury. Arch Surg1995; 134:604-10

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39. J S Barkun, G M Fried, A N Barkun, H H Sigman, E JHinchey, J Garzon, M J Wexler, and J L Meakins.Cholecystectomy without operative cholangiography.Implications for common bile duct injury and retainedcommon bile duct stones. Ann Surg. 1993 September;218(3): 371–379

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INTRODUCTION

The completion of the Human Genome Project (1,2)in 2003, signified a turning point in the field of biosciencesand development of high-throughput technologies. It pavedthe way for numerous innovations and many scientificadvancements of the Post Genome Era in biological sciences.During the course of the project, the presence of sequencevariations led to the inclusion in the last part of their lastfive-year plan to identify and map these variations. Withtheir occurrence roughly every 1000 base pairs, singlenucleotide polymorphisms (SNPs) are among the mostcommon genetic variations (3).

The National Centre for Biotechnology Information(NCBI), a division of the National Library of Medicine(NLM) at the National Institutes of Health (NIH) in U.S.has been actively involved in the genome sequencing efforts

PHARMACOGENOMICS – A NEW PERSPECTIVE IN CLINICAL HEALTHCARES. Karthika a, PK. Ragunath a

ABSTRACTThe post-genome era has heralded the beginning of

incorporating genome-wide approaches in processesbeginning from drug discovery to clinical prescription withthe ultimate aim of viewing modern medicine aspersonalized molecular medicine. A pioneering step in thisdirection is the development of Pharmacogenomics – thestudy of the interplay of genotype and drug efficacy thatseeks to identify the variant genes affecting the response todrugs in individual patients. These variations can be usedto predict whether a patient will have a good response to adrug, a bad response to a drug, or no response at all.Pharmacogenomic studies encompass the sum of allgenes, i.e., the genome with respect to its its impact onpharmacokinetics and pharmacodynamics. Initiallytermed as Pharmacogenetics, it is now synonymous withPharmacogenomics.

A classical example cited in literature is the existenceof a polymorphism in the metabolism of the antituberculousdrug – Isoniazid (INH) that affects the catalytic activity ofthe acetylating enzyme, thus leading to slow acetylation ofINH. Certain populations of the world are associated withthis polymorphism making them slow acetylators. Similarpolymorphisms have also now been studied in many othergenes of pharmacogenomic significance.

The use of non-automated and automated methods forprofiling genotypes has tremendously helped to reveal the


Dr. S. KARTHIKADepartment of BioinformaticsSri Ramachandra UniversityPorur, Chennai - 600 116email : [email protected] / [email protected] Dept. of Bioinformatics

association of single nucleotide polymorphisms, with theircorresponding phenotype.

Apart from the pharmaceutical impact, anothersignificant role of pharmacogenomics has been found inpreventive medicine. It also suggests restructuring the currentdesign of clinical trials by considering the relevant geneticvariations that may considerably affect the trial outcome.Critical clinical decisions will also be benefitted by theunderstanding of the genotype-phenotype associations. Thusit has a promising role to play in diverse areas of clinicalhealthcare including diagnostics, therapeutics, diseaseprevention and target-based drug discovery.

Pharmacogenomics - the rational genomics-basedtherapeutic approach requires several important issues tobe addressed such as the quantum of information that canbe and needs to be divulged to the patient, clinicianacceptance and critical ethical and legal questions.

This review gives a brief insight into this developingfield that has a vast potential in clinical research andhealthcare. A pharmacogenomic approach is a vision forthe future where personalized medicine will be focused onthe patient who has a disease and not just looking at thedisease per se.

Key words: Single nucleotide polymorphism,pharmacogenomics, pharmacokinetics

and is now a popular resource for molecular biologyinformation (1). It played a key role in launching an initiativetowards identifying and mapping the SNPs which culminatedin the NCBI establishing the Single NucleotidePolymorphism database (4) (http://www.ncbi.nlm. nih.gov/SNP) in collaboration with the National Human GenomeResearch Institute (NHGRI). This has now become aninternational effort and many research institutions in variouscountries are involved in this task. Currently, many SNPdatabases exist that can be accessed on the internet. Eg. Asimilar Japanese population based SNP database has alsobeen created (http://snp.ims.u-tokyo.ac.jp/) (5).

Role of Polymorphisms in Pharmacogenomics

Genetic polymorphisms contribute greatly to the inter-individual variations observed in complex humanphenotypes, such as disease susceptibility and our responsesto drugs or environmental chemicals (6). Hence,understanding these variations will assist in unraveling therelationship of individual/population genotype(s) with theresponse to therapeutics. The study of association betweengenetics and drug response is called pharmacogenomics.The potential role of genomics and pharmacogenomics inclinical research and medical healthcare is in modifyingthe existing disease management strategies that will

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additionally incorporate the knowledge of geneticpolymorphisms in drug disposition and effects. This willmake a significant contribution to the pursuit of developingbetter, safer drugs as a part of personalized drug therapies inan era of personalized medicine where the right drug willbe given to the right patient (7,8). Pharmacogenomics isthus, a science that examines the inherited variations ingenes that dictate drug response and explores the ways thesevariations can help predict the outcome of administeringtherapeutic medications as good, bad or no response to thedrug(s).

This is closely related to Pharmacogenetics which hasbeen mainly concerned with the study of the genetics ofdrug metabolism and genetic factors affecting drug targetssuch as receptors. Presently, these terms are usedsynonymously.

Trends: Past and PresentMany phenotypic differences in drug absorption,

distribution, metabolism and elimination (ADME) are partlya reflection of polymorphisms in genes encoding drugmetabolizing enzymes, drug transporters, and/or drug targets(e.g., receptors, enzymes) (9). Initial pharmacogenetic studiesfocused on monogenic traits where these variations affecteddrug metabolism. Current studies look at high-throughputtechniques that reveal the significance of many candidateproteins, influencing pharmacokinetics andpharmacodynamics of a drug – the former involvingmechanisms that influence the concentration of a drugreaching its target(s) and the latter, the drug targetitself (10).

For many drugs, the difference in patterns of patientresponse has been attributed to variations inpharmacokinetic rather than pharmacodynamics. Butrecently, it seems that pharmacodynamic variability isusually more pronounced than in pharmacokinetics. Someexamples of the role of pharmacogenomics onpharmacokinetics are cytochrome P-450 isoenzymes,dihydropyrimidine dehydrogenase, and thiopurinemethyltransferase; some examples of pharmacogenomicsrevealing variations in pharmacodynamics involvecholesteryl ester transfer protein, angiotensin-convertingenzyme, and serotonin transporter (11). Genes ofpharmacogenomic significance can be classified as 1)polymorphisms affecting metabolism of drugs, 2) affectingdrug transport and disposition and 3) polymorphismsaffecting drug targets. The first category includescytochrome P450s which is involved metabolizing drugssuch as Nicotine, Warfarin, Omeprazole, Cyclosporineetc.), N-acetyltransferase -2 ( associated drug – INH) andThiopurine S-methyltransferase ( drug metabolized - 6Mercaptopurin, 6-Thioguanine and Azathioprine).Polymorphisms affecting drug transporters such as MDR-1( Multidrug Resistance Protein ) affect digoxin transport.Individuals homozygous for a particular variant with a highMDR-1 gene expression had lower levels of plasma digoxinlevels following oral administration of the drug.

Polymorphisms affecting drug targets such as dopamineD3receptor affects response to some antipsychotic drugs, thosein angiotensin converting enzyme influence the responseto ACE inhibitors and polymorphisms in cholesteryl estertransfer protein modify response to cholesterol loweringstatin – Pravastatin.( 12)

The majority of drug responses involve many proteinsand their corresponding genes could have severalpolymorphisms making it unlikely that a singlepolymorphism in a single gene could result in a high degreeof drug response variability in a consistent fashion. Thus,genomic approaches seem more appropriate to study thecontribution of polymorphisms of several genes involved ina particular drug response. Such approaches topharmacogenomics have shown promise in relating drugtarget polymorphisms to response or toxicity, andincorporating this strategy in drug discovery and developmentis a new approach that is being used by many pharmaceuticalcompanies (13). Two such FDA approvedpharmacogenomics-based drugs available currently in themarket are Herceptin® (Genentech) and Velcade®

(Milleneum). Herceptin® (Trastuzumab) is indicated fortreatment of HER2 protein overexpressing, metastatic breastcancer. Velcade® (Bortezomib), a proteasome inhibitor isindicated for treatment of Multiple Myeloma.

A combination of pharmacogenomics and personalizedmedicine will eventually change the method of current drugprescription to one where drug selection and dosage processfor any given patient will be advanced by genomic knowledgeand information gained by these strategies (14).

Metabolic phenotyping studies can be done byadministering a probe drug or substrate and measuring themetabolites with the clinical outcomes if any. Thedisadvantage of this approach is that it tends to be laborintensive and requires repeated sample collection from theindividual or patient, being tested. On the other hand,genotyping allows determination of individual DNAsequence variants associated with particular traits. DNAsample is commonly obtained from whole blood cells andbuccal cells. Genomic DNA is isolated from from othercellular material. After a specific region of interest isidentified and amplified, using polymerase chain reaction(PCR). Subsequently, gel electrophoresis is often performedto verify that PCR was successful and that the amplifiedtarget sequence is the correct size. . Some of the commonlyused genotyping methods include gel electrophoresis-basedtechniques, such as polymerase chain reaction (PCR)coupled with restriction fragment length polymorphismanalysis (RFLP) does not rely on automated technologyand is practical for laboratories genotyping a limited numberof samples. Other gel electrophoresis-based techniquesinclude multiplex PCR, and allele-specific amplification.Automated genotyping methods include Pyrosequencing,in which the principal allele discrimination method is aprimer extension reaction coupled with a luciferase-basedenzyme reaction. TaqMan uses fluorescence-labeled probes,

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in addition to PCR primers, in the reaction mixture,enabling PCR amplification and allele discrimination inthe same step. Mass spectrometry differentiates DNAmolecules based on their mass. Denaturing high-performance liquid chromatography (DHPLC) using areverse-phase ion-pair column, helps discriminate betweenvariant and nonvariant. Some otther fluorescent dye-basedhigh-throughput genotyping procedures include includingoligonucleotide ligation assay and direct heterozygotesequencing. The latest advances in genotyping includeMicroarrays. (15,16)

The development of a new related field that servesto complement pharmacogenomics is that ofpharmacoproteomics. The combined effort will endeavourto enhance the development of personalized medicines. Thehigh-throughput techniques in pharmacoproteomics have apotential contribution in the area of molecular diagnosticsthat forms an essential component of personalized medicine.As the functional impact of genetic variations is seen in thefunctioning of the encoded protein, pharmacoproteomicswill explain the functional phenotypic consequence of thepatient-to-patient variation, to a greater extent than thatoffered by the genotype alone. Another role foreseen is incharacterization of multifactorial diseases where it can helpmatch a particular target-based therapy to a particular markerin a subgroup of patients (17).

An innovative and exciting prospect, this specializationof genomics has an immense potential in successfuldevelopment of better, effective therapeutics as it will playa role in overcoming important setbacks in the traditionalprocess of drug discovery. Genomics and its derivative fieldsalong with their high throughput technologies promise toinfluence different stages of drug discovery process such astarget selection, lead identification, lead optimization, thusyielding safer and effective drug candidates (18).

Role of Pharmacogenomics• In Disease Management & Therapeutics – Some recentexamples:

Many biological systems and their diseases have beenthe focus of pharmacogenomic studies with a potentialimpact in areas ranging from screening, diagnosis, monitoringtherapy response, choice of drugs etc. Some highlightingexamples in this regard include diseases such as AcuteLymphoblastic Lymphoma, Asthma, Gastric Cancer,Multiple Sclerosis and Osteoporosis.

Acute Lymphoblastic LeukemiaThe childhood-onset disease Acute Lymphoblastic

Leukemia is a notable case where pharmacogenomics modelshave been suggested which could aim at providing maximumefficacy with minimum adverse effects of existing therapeuticmedications when prescribed using the patient genotype withfavourable response and low toxicity, or to identify noveltherapeutic targets in lymphoblasts that are resistant toconventional antileukemic drugs (19). These drug targetspossessing polymorphisms that favour optimum drug

response with low toxicity and lesser chances of relapsewill be ideal for developing newer, safer and more effectivechemotherapeutic agents for cancer therapy. ALL is alsoperceived to serve as a model for similar pharmacogenomicapplication in other human cancers. Early treatment responseinvolving measurement of minimal residual disease reflects,both drug responsiveness of leukemic cells and hostpharmacodynamics/pharmacogenomics, has been consideredthe most reliable prognostic indicator for gauging the intensityof treatment. (20).

AsthmaBeta2-agonists are the most effective bronchodilators

available providing rapid relief of asthma symptoms andthe degree of the drug response varies greatly between patientsand genetic factors have a major role in it (21).Pharmacogenomic analysis of the commonly used anti-asthmatic agents such as ß-agonists, leukotriene modifiers,and corticosteroids showed that relative risk–benefit ratio ofa particular therapeutic course for an individual patient couldbe identified by using “panels” of polymorphisms. Targetedtherapy will thus help provide optimum benefit for asthmapatients who respond preferentially to certain medicationsand avoid toxicity by prescribing drugs after assessing thelikelihood of adverse drug reactions (22).

Gastric CancerTherapeutics based on the genotyping of each patient

is also predicted as the future goal of personalized medicine(or tailor-made medicine) for chemotherapeutic managementof advanced gastric cancer with drugs such as 5-fluorouracil,Paclitaxel, Docetaxel etc that are associated with side effectsand drug resistance. These drugs play a role in managementof advanced gastric cancer and complements surgicalmanagement of one of the common malignancies in theworld (23).

Multiple Sclerosis & OsteoporosisAnother area of potential impact is foreseen in diseases

that presently, have no definitive treatment eg. theneurodegenerative disease - Multiple Sclerosis (24). anddiseases where therapy is typically required for several yearsbefore outcomes can be evaluated for an individual eg. thesystemic skeletal disease – Osteoporosis (25). These arebeing investigated currently.

• Understanding Disease Mechanisms of BiologicalSystems

There has been a significant increase in understandingof biological systems and their disease mechanisms eg.the cardiovascular system. Pharmacogenomic approachesare emerging for many medications used in differentcardiovascular conditions that would assist clinicians indecision-making being more precise to optimize thebenefit-to-risk ratio. When combined with molecularimaging, molecular pathway mechanisms can be studiedthat will allow us to monitor health and disease andenable the enormous shift from genetic to genomicmedicine (26).

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• Critical Clinical DecisionsThe use of the anticoagulant Warfarin, is difficult due

to the variations in the dose required to have a therapeuticeffect, and the adverse effect of serious bleeding. The druginterferes with the recycling of vitamin K in the liver whichreduces the activation of several clotting factors. Many genesseem to be involved in the biotransformation and mode ofaction of this drug and the outcome of large ongoing studiesof these along with assessment of environmental factors,will suggest a method to predict the required warfarin dosewith minimized risk of haemorrhage (27).

Administering anti-fungal drugs in theimmunocompromised state or for refractory/ persistentmycoses is another daunting scenario. Genes with potentialpharmacogenomic significance include a) drug transportersinvolved in absorption and excretion, b) phase I enzymes(e.g., cytochrome P450-dependent mixed-function oxidases)and phase II enzymes (e.g., glucuronosyltransferases)contributing to metabolism, and c) molecules involved indrug distribution (e.g., albumin, A1-acid glycoprotein, andlipoproteins). Tools of population genetics can be used todefine inter-individual variation in drug ADME and yieldpharmacogenomic models for the observed genetic variationsin antifungal pharmacokinetics (28).

Monitoring and management of adverse drug reactionsof chemotherapy in cancer patients is another applicationof Pharmacogenomics. The impact of polymorphism incytidine deaminase that metabolizes the anticancer drug -Gemcitabine was studied in Japanese cancer patients. Aparticular genotype harboring a nonsynonymous SNP,(Ala70Thr) was associated with decreased clearance ofgemcitabine, and increased incidences of neutropenia whenpatients were coadministered platinum-containing drugs orfluorouracil.(29)

Enhanced Diagnostic TestsSeveral technologies used in personalized medicine

include SNP genotyping, haplotyping, gene expressionstudies by biochip/microarrays and proteomics. Moleculardiagnostics will play a key role in personalized medicinewhere therapy and diagnosis will be integrated (30). InJanuary 2005, the US Food and Drug Administration (FDA)granted market approval for the first pharmacogenetic testusing a DNA microarray, the AmpliChip CYP450GeneChip(R) (AmpliChip), which genotypes cytochromeP450 CYP2D6 and CYP2C19 (31). It classifies individualsinto two CYP2C19 phenotypes (extensive metabolizers[EMs] and poor metabolizers [PMs]) by testing three allelesand into four CYP2D6 phenotypes (ultrarapid metabolizers[UMs], EMs, intermediate metabolizers [IMs], and PMs(32). Cytochrome P450 is one of the best studied drug-metabolism associated protein complex in the light ofpharmacogenomics.

The application of this knowledge is relevant to thepopulation type being considered for therapy. Poormetabolizers (PMs), lacking the enzyme, account for up to

7% of Caucasians for CYP2D6 and up to 25% of East Asiansfor CYP2C19. Patients having three or more active CYP2D6alleles; the Ultra Rapid Metabolizers account for up to 29%in North Africa and the Middle East. As CYP2D6 metabolizesmany of the psychiatric drugs, psychiatry has become thepioneer for the practical clinical use of pharmacogenetictesting that has relevance in the usage of drugs such astricyclic antidepressants and antipsychotics (33). Evaluationof pharmacokinetics of citalopram in relation to geneticpolymorphism of CYP2C19 was done in Chinese volunteerswhose genotypes and phenotypes were known beforeadministering the drug and studying its varied effects.Citalopram, a potent and selective serotonin reuptakeinhibitor is used as an antidepressant. It is mainlymetabolized to N-desmethylcitalopram and furtherdemethylated to didesmethylcitalopram, the latter beingcatalyzed by CYP2C19, CYP2D6 and CYP3A4. This studysupported the involvement of CYP2D19 in catalyzing thisreaction and showed the varied genotype of this gene leadsto differences in drug pharmacokinetics when comparingpoor metabolizers (PM) and extensive metabolizers (EM).Some of the findings included moderately higher t1/2 valuesof citalopram in PMs than the EMs. The CLoral (oral clearance) of citalopram in poor metabolizers (17.1 ± 2.0 l/h) wassignificantly lower than that of extensive metabolizers (24.1± 1.6 l/h). PMs showed significantly longer tmax values ofdesmethylcitalopram than in extensive metabolizers (34).CYP testing is also applicable in toxicology and ADMEprofiling to guide drug development. Combined withpharmacotherapy, it can aid in effective decision-makingregarding choice of drugs and dosage (35).

Another landmark test to identify metastatic colorectalcancer patients with indications for use of topoisomerase Iinteractive drug – irinotecan was approved by FDA in 2005.The genetic test (Invader UGT1A1 Molecular Assay),conducted on genomic DNA from peripheral blood,identifies homozygosity for the UGT1A1*28 allele. Suchpatients clear irinotecan and its metabolites more slowlythan the rest of the population, and so have greater exposureto active drug after a standard dose. The FDA-approved testlabel states that “a reduced initial dose should be consideredfor patients known to be homozygous for the UGT1A1*28allele”(36,37).

Role in Clinical TrialsThe post-genome era has led to advances such as high-

resolution SNP maps and technical approaches likemicroarray have enabled incorporating genome-wideassociation studies in clinical trials and can contribute byidentifying disease-susceptibility genes useful as prognosticindicators, process of drug discovery, and selection oftherapy. An SNP map containing genetic variants relevantto drug transport, metabolism, and receptor interaction canbe significant in drug selection and point to conditionswhere careful drug dosage monitoring is required (38).

At the 42nd American Society of Clinical Oncology(ASCO) annual meeting in Atlanta, Eli Lilly and Company,

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a leader in thoracic cancer, unveiled two breast cancer studiesinvolving pharmacogenomics and its chemotherapiesGEMZAR® (Gemcitabine HCl) and ALIMTA® (Pemetrexed).They are among the early trials involving pharmacogenomicsin breast cancer treatment and have so far shown signs ofbeing able to predict response related to both general anddrug- specific chemotherapy and biomarker analysis.

Clinical Data Inc. has launched the Phase III clinicaltrial for the depression drug Vilazodone and concurrentlylooking forward to develop a diagnostic test based on geneticbiomarkers that can predict drug efficacy and response.

Many pharmacogenomics based clinical trials arecurrently underway in different phases of study. Eg. Some atthe US National Institute of Health are mentioned at URL:http://www.clinicaltrials.gov/.

Improved Prospects in Preventive CareIf risk for a given disease is predicted to be high, as

judged by the SNP pattern of a patient, preventive therapyand lifestyle adjustments (diet, exercise, etc) may beimplemented (38). This could help prevent the onslaughtof diseases that can be managed by preventive measuresbefore the onset of the actual disease.

Current DatabasesPharmGKB - The Pharmacogenetics and

Pharmacogenomics Knowledge Base, is a publicly availableInternet research tool developed by Stanford University withfunding from the National Institutes of Health (NIH) and ispart of the NIH Pharmacogenetics Research Network(PGRN), a nationwide collaborative research consortium.Its aim is to aid researchers in understanding how geneticvariation among individuals contributes to differences inreactions to drugs. The database is a central repository forgenetic, genomic, molecular and cellular phenotype dataand clinical information about people who have participatedin pharmacogenomics research studies. The data includes,but is not limited to, clinical and basic pharmacokineticand pharmacogenomic research in the cardiovascular,pulmonary, cancer, pathways, metabolic and transporterdomains (39) available at URL: http://www.pharmgkb.org/

Human Membrane Transporter Database - for DrugTransport Studies and Pharmacogenomics is another relateddatabase which currently contains data on more than 250human membrane transporters and related proteins, theirstructure, function, sequence variants, and substrates,especially drugs. As this database is intended to supportpharmacogenomic studies, it also provides information onsequence variants, altered functions caused bypolymorphisms/mutations, and the (patho) physiological roleand associated disease (40) available at URL: http://lab.digibench.net/transporter/.

Limitations and ObstaclesThough found to influence many clinical situations,

there are others where it may not be very useful. An examplefor this is the assessment of variations in metabolism ofand clinical response to many commonly prescribed non-

steroidal anti-inflammatory drugs (NSAIDs), thoughcytochrome activity plays an essential role in the drugmetabolism (CYP2C8/9) (41). The market withdrawals ofrofecoxib (Vioxx) and valdecoxib (Bextra) led to great interestin assessing the side effect profiles of cyclooxygenase (COX)inhibitors. CYP2C9 genotype is considered a risk factor asmany COX inhibitors are CYP2C9 substrates in vitro. Butthe study showed that apart from the effect of CYP2C9genotype, a major determinant of clearance of this categoryof drugs, it was also felt necessary to consider CYP2C8genotype for some of them and, possibly, CYP3A4 activityfor some others (42). Hence prescribing these over-the-counter drugs with the relevant genotypic information maynot be easily accepted by the clinicians and patients alikeas some of them are prescribed for simple symptomaticrelief.

There are inherent obstacles that have to be surmountedfor the widespread use of pharmacogenetics and includedifficulties such as economic feasibility and awarenessamong medical practitioners regarding the application andinterpretation of results (31, 43).

Apart from educating the healthcare personnel andassessment of questions in ethical and legal concepts, publicawareness about consent and implications of genetic testingin drug therapy and disease management is also a pressingneed (44). This would aid interpretation of risk assessmenttests where a predisposition to a pathological condition maynot mean actual suffering from the disease. Adequatepreventive measures can be instituted where tests indicate ahigher probability of disease in later years, ultimatelypreventing many illnesses. But the burden of the informationand fear may be unintended outcomes that also have to beweighed carefully.

CONCLUSIONThe traditional approach of one-drug-fits-all does not

cater to the need of smaller populations/subpopulations thatshow a similar phenotypic profile with the others but havean underlying distinct genetic signature that consequentlyleads to variation in drug response.

Pharmacogenomics combining the potential ofgenomics with pharmacology has tremendous practicalpotential to significantly enhance the ability of cliniciansto use medications in a safe and effective manner (13). Thefuture is not far away when these will be a part of thepersonalized drug prescription and estimates foresee this bythe year 2015, according to the lay journal Time(45) and2020, by JAMA(46).

An overall decrease in the number of adverse drugreactions, the number of failed drug trials, the time takenfor approval of a drug, the duration of medicationadministered, the number of medications taken by patientsbefore eventually finding the effective therapy, thepathological effects of a disease (through early detection),and a simultaneous increase in the range of possible drugtargets will promote a net decrease in the cost and improvethe quality of health care.

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14. Vizirianakis IS. Improving pharmacotherapy outcomesby pharmacogenomics from expectation to reality.Pharmacogenomics 2005;6(7):701-11.

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16. Aquilante CL, Zineh I, Beitelshees AL, Langaee TY.Common laboratory methods in pharmacogenomics studies.Am J Health Syst Pharm 2006 Nov 1 ;63(21) :2101-10.

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19. Kager L, Evans WE. Pharmacogenomics of acutelymphoblastic leukemia. Curr Opin Hematol2006;13(4):260-5.

20. Pui CH. Recent advances in childhood acutelymphoblastic leukemia. J Formos Med Assoc2004;103(2):85-95.

21. Bhatnagar P, Guleria R, Kukreti. Pharmacogenomicsof beta2 agonist key focus on signaling pathways.Pharmacogenomics 2006;7(6):919-33.

22. Wechsler ME, Israel E. How pharmacogenomics willplay a role in the management of asthma. American Journalof Respiratory and Critical Care Medicine 2005;172:12-8.

23. Katoh M, Katoh M. Pharmacogenomics on gastriccancer. Cancer Biol Ther 2004;3(6):566-7.

24. Annibali V, Ristori G, Cannoni S, Romano S, ViscontiA et al. Multiple sclerosis pharmacogenomics andpersonalised drug treatment. Neurol Sci 2006;27(5):347-9.

25. Nguyen TV, Eisman JA. Pharmacogenomics ofosteoporosis opportunities and challenges. MusculoskeletNeuronal Interact 2006;6(1):62-72.

26. Ginsburg GS, Donahue MP, Newby LK. Prospects forpersonalized cardiovascular medicine the impact ofgenomics. J Am Coll Cardiol 2005;46(9):1615-27.

27. Wadelius M, Pirmohamed M. Pharmacogenetics ofwarfarin current status and future challenges.Pharmacogenomics 2006. ( Epub ahead of print)

28. Meletiadis J, Chanock S, Walsh J. Humanpharmacogenomic variations and their implications forantifungal efficacy. Clin Microbiol Rev 2006;19(4):763-87.

29. Sugiyama E, Kaniwa N, Kim SR, Hasegawa R, MaekawaK, Saito Y. Pharmacokinetics of gemcitabine in japanesecancer patients: the impact of a cytidine deaminasepolymorphism. Journal of Clinical Oncology. 2007 Jan1;25(1):32-42.

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33. de Leon J, Armstrong SC. Clinical guidelines forpsychiatrists for the use of pharmacogenetic testing forCYP450 2D6 and CYP450 2C19. Cozza KL Psychosomatics2006;47(1):75-85.

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3. Clinical Data. CO Genics, PGx Health; Available from:URL: http://www.clda.com

4. St. Jude Children’s Research Hospital. 2004 May 27;Available from: URL: http://www.stjude.org/media/0,2561,453_5297_11247,00.html

5. Personalised Medicine 2004 ; Available from URL:www.leaddiscovery.co.uk/reports/personalised

Review Article


PSYCHOLOGICAL AUTOPSY: the Psychological Assessment of an equivocal DeathP. Sampath kumar a, Joshua Ashok a, Srikanth Sankar a, S.J. Aswin Sayiram a, Anand Kumar Vasudevan b

ABSTRACTThere are times when physical evidence and evidence

found at autopsy does not reveal the cause and mode ofdeath. This is known as equivocal death.. The psychologicalautopsy is a retrospective construction of a decedent’s lifeinitiated to get a better understanding of his death. It isused to determine the victim’s psychological intent, usinginterviews and examination of documents to reconstruct thebehaviour, personality, lifestyle, habits and history of thevictim prior to death. Psychological autopsy aids as aninvestigative tool which is at the outer edge of professionalknowledge and practice in that it requires an application ofskills, experience, and training to assess a variety of factorsincluding the behaviour, thoughts, feelings, and relationshipsof an individual who is deceased. There may be issues thatthe deceased or his or her family does not want facts to berevealed that require special handling. Hence the interviewsof family members, friends, and relatives may be adequate.So the interviewer has to be flexible The interviewer shouldestablish mutual respect and confidence, with the informant,and ensure confidentiality and anonymity, and also obtainan informed consent before the interview. Hence one shouldbe qualified and skilled to conduct the interview. Falseinformation also can be given due to lack of memory or it


Prof. P. SAMPATH KUMARProfessor & HeadDept. of Forensic MedicineSri Ramachandra Medical College and Research Institute,Porur, Chennai - 600 116.email : [email protected] Dept. of Forensic Medicine, Sri Ramachandra Universityb University of Melbourne, Australia

may be intentional. Suicide note, Personal documents,Medical records, School records, Military records,Employment records, should be carefully analysed. Withthe above mentioned information, a psychological autopsyreport is produced, the final conclusion depends on theaccuracy of the data collected from the interviews,examination of relevant documents and other materials.Therefore the interviewee’s probabilities and limitation toscience should be noted .Thus the final judgement as tothe mode of death is based upon a review of all the knownfacts and circumstances; including the coroner’s report,forensic medical report, police reports, crime scene analystreports, and the psychological reconstruction ,so that peoplemay learn from the tragedy and, hopefully, be cautious andreduce the chances of similar occurrence in future.Psychological autopsy is most often used in cases ofsuspected suicide or homicide in an attempt to reconstructthe personal life and character of the deceased, to uncoverhidden secrets that may help to give family members peaceof mind and also plays a role in many legal suits,malpractice suits, insurance claims, and criminalinvestigations.MesH words : Cause of death, autopsy, coroners andmedical examiners.

“Human consciousness is an unusual template of experienceand emotions”

IntroductionIn every instance of death, a physician must distinguish

both the cause and mode of death. The cause of death isdefined as “the original underlying medical condition whichinitiates the lethal chain of events culminating in death”1.It is the duty of the medical examiner, coroner, and lawenforcing authorities, to determine the mode of death in allviolent and suspicious death investigations. There are timeswhen physical evidence and evidence found at autopsydoes not reveal the mode of death. This is known asequivocal death. Equivocal deaths often involve questionsthat surface around suicide, homicide, accidental or somenatural deaths. Those deaths that fall under the category of“undetermined” are based on what is found at the scene ofcrime and autopsy, but which frequently require a closer“psychological” investigation and examination. The

psychological autopsy was developed as a further post morteminvestigative tool that aids in the determination of theperson’s death. The psychological autopsy is a retrospectiveconstruction of a decedent’s life initiated to get a betterunderstanding of his death. It is used to determine thevictim’s psychological intent, using interviews andexamination of documents to reconstruct the behaviour,personality, lifestyle, habits and history of the victim priorto death2. The concept and technique of the psychologicalautopsy was developed by Dr. Edwin S. Shneidman whodefined the psychological autopsy as:

“A behavioural scientific impartial investigationof the psychological (motivational, intentional) aspectsof a particular death. It legitimately conducts interviews(with a variety of people who knew the decedent) andexamines personal documents (suicidal notes, diaries,and letters) and other materials (including autopsyand police reports) that are relevant to the role in theindividual’s death”3.

Behind the scene of an equivocal death investigation

Role of the medical examiner in the investigation:The medical examiner, who is the forensic pathologist

defines the cause of death, provides details about themanner and circumstances of death and the results of amedico-legal autopsy. The prime function of the medicalexaminer is medico-legal certification of the manner ofdeath, which has important legal, social, medical andresearch implications4.

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Role of the investigators: An investigator in the death team investigation includesthe magistrate / coroner, medical examiner, psychologist,psychiatrist, a psychiatric social worker, or a policeinvestigating officer and other law enforcement authorities,as psychological autopsy aids as an investigative tool whichis at the outer edge of professional knowledge and practicein that it requires an application of skills, experience, andtraining to assess a variety of factors including the behaviour,thoughts, feelings, and relationships of an individual who isdeceased 4.Therefore, the interview with the family, friends,co-worker, relatives, neighbours, physicians and otheracquaintances to prepare a psychological autopsy report arecarried out mainly by the mental health professionals andbehavioural science investigators.

Primary goals of these mental health professionals andbehavioural scientists are 5:a) To determine the mode of death.b) Reasons for death at that particular period of time.c) Assessment of lethality (suicide).d) Psychotherapeutic value to the survivors.

Protocol to conduct an equivocal death interviewa) Ethical considerations concerning the interview, is ofprior importance, the integrity of the deceased must berespected (Young, 1992). There may be issues that thedeceased or his or her family does not want revealed orfacts that requires special handling.

b) History of the actual events must be reviewed, laterthe interviews of family members, friends, relatives,colleagues at work/school, neighbours, physicians, priest,acquaintance and eyewitnesses, should be conducted.

c) Approaching the informant to conduct an interview isan important consideration, the informants are contactedby mail and later a phone call, to avoid contact refusalrate 6. So the interviewer has to be flexible and ready to re-schedule the interview.

d) The interviewer should establish mutual respect andconfidence, with the informant, and ensure confidentialityand anonymity, and also obtain an informed consent beforethe investigation.7

e) The skills of interviewing includes, proper language,clarity, listening more than speaking, no threatening questions,no repetitions, avoid loaded questioning and more than oneinformant in a single interview (College of Health,1994).

f) The interviewee may have motives of giving exaggeratedinformation or concealing facts, or give pertinent informationto protect the image of the victim and family. Falseinformation also can be given due to lack of memory or itmay be intentional. Therefore it is important to explain tothe interviewee the significance of the information he / sheare providing. It is the role of the interviewer to be able toassess distorted or irrelevant information.8

Salient features in an equivocal death interview: 9

1) Description of the deceased: the personal views aboutthe deceased.

2) Period of association with the deceased: how long theyknow the deceased, how often they see each other, type ofrelationship between them.

3) Any changes noticed in behaviour or emotional distressassociated with the deceased.

4) Any problems noticed by the interviewee, or discussedwith deceased.

5) Observed or expressed mental status of the deceasedto situation of depression and stress.

6) Recent changes physically observed: pain, signs ofillness, fatigue, tension, or loss of appetite, changes in sleeppattern, insomnia, wakes up throughout the night.

7) The interviewee’s reasons behind the death: whatwould have probably happened and why.

Other sources:10

• Suicide note: This play an important role to solve thewhole issue if it is proved that the deceased had written thesuicide note (verified by a forensic document examiner),the contents, language (specific references to suicide ormorbid content). Suicide note also plays as an experimentalcontrol for the mental health professionals in interviewingprocess, when they are not disclosed about the evidence,and to derive their opinion to provide an estimate ofvalidity 10.

• Personal documents: Letters (family, friend, relatives,or acquaintances), dairies, videos, and literature read recentlyand in past (morbid content), e mail, threats notes ormessages received recently, bills, tickets, and pornographiccollections if any.

• Medical records: Visits to physicians, medical illness,family history of illness, whether under medications.

• School records: Information such as change in academicperformance or absenteeism, conduct and character ingeneral

• Military records: Reveal education and trainingbackground, areas of deployment, promotions, efficiencyand obsession for weapons.

• Employment records: Performance, conduct, alibi ofwork and absenteeism.

With the above mentioned information, apsychological autopsy report is produced, and laterreviewed by the death investigation team to determinethe mode of death11. The psychological autopsy isconsidered ultimately to be an expert opinion; thereforeit depends on the accuracy of the data collected from theinterviews, examination of relevant documents and othermaterials. Therefore the interviewee’s probabilities andlimitation to science should be noted 2.

Short Review


Evaluation of psychological autopsy report The psychological autopsy report provides detailedinformation about the death using various sources includingthe autopsy report, medical records, relevant documents andinformation gathered from interviews with key informants.These sources and information provided is to be clearlydocumented for evaluation and its potential validity. Thebehavioural scientist and other mental health professionalswould be expected to provide more systematic details aboutthe important psychological stages in the person’s thoughtprocesses e.g. motivation and personality, to deliver a formalevidence for the conclusion. Actually there is no welldeveloped conceptual or theoretical basis for derivingconclusions from various sources of information, due tolack of standardised technique or specific procedure inconducting the psychological autopsy there is an area ofpotential weakness of this procedure. Here, the investigatoris merely interpreting a prior set of facts rather than predictingfuture behaviour based upon the limited facts availablebefore assessment. The final judgement as to the mode ofdeath is based upon a review of all the know facts andcircumstances; including the coroner’s report, forensicmedical report, police reports, crime scene analyst reports,and the psychological reconstruction3. Finally, to analyseand conclude suspicious deaths or ambiguous fatalities, andto facilitate the expansion of knowledge so that people maylearn from the tragedy and, hopefully, be cautious and reducethe chances of similar occurrence in future.

Our Experiences an example:-A 23 year male was rushed to the causality with

bleeding injury abdomen, with strong smell of alcohol, withalleged history of tripping and falling over an sharp vegetablecutting weapon in an inebriated condition. He maintainedthe same history up to his death. By practising our guidelinesof psychological autopsy the investigation revealed that therewas a scuffle between two brothers with a double edgedweapon, which lead to his death. Hence the opinion as toaccidental injury was changed as homicidal.

Surgically sutured wound on the lateral side of the abdomenshowing the homicidal injury

ConclusionPsychological autopsy is most often used in cases of

suspected suicide or homicide in an attempt to reconstruct

the personal life and character of the deceased, to revealthat may help to give family members peace of mind andalso plays a role in many legal suits, malpractice suits, andinsurance claims, including criminal investigations.Psychological autopsy has become a valuable tool in theinvestigation and, at times, resolution of questionable andequivocal death cases. Despite the weakness of the evidenceand procedures used in this technique, mental healthprofessionals face problems while reconstructing apsychological autopsy in both civil and criminal matters,when questions can be raised regarding the mental state ofthe deceased prior to death. Therefore, it is importantto establish the value that the report may add to theproceedings, and that includes the admissibility ofpsychological autopsies as evidence in court hearing whichwill be in the near future.

BIBLIOGRAPHY1. Spitz, W.U. (1993). Medicolegal investigation ofdeath, 3rd edn. Charles C. Thomas, Springfield: Chapter 7,pg125-127.

2. Litman, R.E. (1989). 500 psychological autopsies.Journal of forensic Sciences 34: pg 638-646.

3. Shneidman, E.S. (1994). The psychological autopsy.American Psychologist Vol 49: pg 75-76.

4. Jobes & Berman, A.L (1986). “The impact ofpsychological autopsies on medical examiner’s determinationof manner of death” Journal of forensic Sciences, Jan 1986,Vol.31, pg 177-189.

5. Davies, J.A, Emer, D.M, (2003). “Role of and techniquein forensic psychology” Forensic medicine: Clinical andpathological aspects, Chapter 46, pg 705-721.

6. Brent, D., Perper, J., Kolko, D. (1998). “Thepsychological autopsy: methodological considerations forthe study of adolescent suicide” Journal of AmericanAcademic Child and Adolescent Psychiatry Vol 27, pg 307-323.

7. British Psychological Society, (1990). “Ethical aspectsof psychological autopsy. Acta Psychiatrica ScandinavicaVol 84, pg 482-487.

8. Hawton, K., Appleby, L., Platt S., Foster, T. (1998).“The psychological autopsy approach to studying suicide”:a review of methodological issues. Journal of AffectiveDisorders 50, pg 269-276.

9. Davis, J. (1993). The use of Behavioural scientists inlaw enforcement. The Law Enforcement Quarterly. J Davis,San Diego District Attorney’s office Vol 27, pg 345-367.

10. Otto, R.K. (1993). An empirical study of the reports ofAPA’s peer review panel in the congressional review of theUSS lowa incident. Journal of Personality Assessment Vol61, p 425-442.

11. David C, Cremer M, (2000) “The psychologicalautopsy”: The pathology of trauma. Arnold, London. Chapter30 pg 475-483.

Short Review


PULSE THERAPY IN DERMATOLOGYRashmi Mittal a, Sudha R a, Murugan S a, Adikrishnan a, Shobana S a, Anandan S a

INTRODUCTION

Pulse therapy means the administration of large(suprapharmacologic) doses of drugs in an intermittentmanner to enhance the therapeutic effect and reduce theside effects 1.

PULSE CORTICOSTEROID THERAPY

The first reported use of pulse administration ofcorticosteroids is attributed to Kountz and Cohn who usedit to successfully prevent renal graft rejection 2. Thereafter,pulse doses of corticosteroids were used for other diseasessuch as lupus nephritis, rheumatoid arthritis and pyodermagangrenosum 3, 4

The introduction of dexamethasone cyclophosphamidepulse (DCP) therapy for a blistering disorder, pemphigusvulgaris, by Pasricha et al has revolutionized the therapy forpemphigus 5. The therapy has since been used to treat alarge number of patients at several centers in India 6 andabroad 7. The dermatological disorders in which corticosteroidpulse therapy has been advocated are pemphigus vulgaris,an autoimmune blistering disorder, systemic sclerosis,systemic lupus erythematosus, dermatomyositis, pyodermagangrenosum, toxic epidermal necrolysis, Steven Johnson’ssyndrome, lichen planus, alopecia areata, sarcoidosis, andsystemic vasculitis. Pulse corticosteroid therapy has beenproposed as a means of rapidly controlling life-threateningor serious conditions with minimal toxicity, allowing forless aggressive long term maintenance therapy. The selectionof drugs and their dosages has been completely arbitary andbased upon intuition and convenience. Conventionally,methyl predinsolone was the agent most commonly used.The choice of dexamethasone made the treatmentconsiderably more affordable and accessible to patients.

ABSTRACTPulse therapy means the administration of large

(suprapharmacologic) doses of drugs in an intermittentmanner to enhance therapeutic effects and reduce adverseeffects. Dexamethasone and cyclophosphamide in bolusdoses have been widely used in pulse form to treat variousdermatological disorders. Pulse corticosteroids have alsobeen tried in other conditions like renal graft rejection, lupus

nephritis and other autoimmune disorders. This form oftherapy has given excellent treatment response with veryfew side effects. Corticosteroids, immunosuppressives,antifungals, antivirals are the commonly used drugsadministered in pulse doses.

Key words: Dexamethasone, pulse drug therapy,cyclophosphamide, autoimmune diseases.

Short Review


Dr. RASHMI MITTALAssistant ProfessorDepartment of Dermatology & STDSri Ramachandra Medical College and Research InstitutePorur, Chennai – 600116.E-mail : [email protected] Department of Dermatology

There was concern among some workers about theequivalence of 1000mg of methylprednisolone and 100mgof dexamethasone and some groups have administeredpulses of 136mg of dexamethasone. However , a dose of1000mg of methylprednisolone is as arbitary as a dose of100mg of dexamethasone and in the absence of evidencethat 136mg pulses of dexamethasone are more effective nearlyall centres continue to use 100mg boluses.8 Moreover, thecost of dexamethasone cyclophosphamide pulse therapyworks out to be 1/3rd of the total cost of methylprednisolonepulse.

Dexamethasone Cyclophosphamide Pulse (DCP)therapy - divided into 4 phases.

Phase I consists of Dexamethasone 100 mg in 5%Dextrose as a slow IV infusion over 2 hours for threeconsecutive days along with cyclosphosphamide 500 mginfusion on one of the days. This constitutes one DCP. SuchDCPs are repeated every 28 days till no new lesions appearbetween pulses. Cyclosphosphamide 50 mg / day is givenorally. During this phase the patient may continue to developrecurrences of clinical lesions in between the DCPs andcan therefore be given conventional doses of oralcorticosteroids to achieve quicker clinical recovery. Afterthe skin and mucous membrane lesions have subsidedcompletely and the additional medication withdrawn , thepatient is considered to have entered phase II9.

Phase II consists of the DCP schedule given for a fixedduration of 9 months.

Phase III , only oral Cyclophosphamide 50 mg / day isgiven for 1 year

Phase IV, all the drugs are with drawn and the patientis followed up.

There was initial alarm and anxiety about the large dosesof corticosteroids and cyclophosphamide, but today thistherapy is given as a routine infusion, often in a day care orOPD setting, with the patient going home a few hours aftercompletion of the infusion.

There have been modifications of DCP therapy10.Cyclophosphamide is known to cause oligo / azoospermiaand amenorrhoea. For unmarried patients who have not


completed their family, cyclophosphamide is replacedwith azathioprine 50 mg (DAP therapy). Methotrexate,7.5 mg (DMP) was instituted in patients who were notcompleting Phase I even after 12 pulses (1 year) of DCPor DAP therapy.

Contraindications of DCP therapyDCP therapy can be given to patients of all ages but

the doses have to be reduced to half for children below theage of 12 years 9. It can also be administered to patientshaving other medical problems like diabetes mellitus,hypertension, osteoporosis, tuberculosis. Diabetic patientsneed to be given 10 units of soluble insulin for every 500ml bottle of 5% dextrose dissolved in the same drip. Inaddition they must be given the routine treatment for diabetesmellitus. Similarly patients having concomitant diseases likehypertension and tuberculosis must receive the respectivemedication. If there is serious infection elsewhere or if thepatient has severe bacterial, viral or fungal infections, thepulse may be delayed for a week or two till the infectionhas been brought under control. Pulse therapy is absolutelycontraindicated in pregnant, lactating and unmarriedpatients.

Laboratory monitoringAs a routine, it is mandatory to admit every patient

enrolled for pulse therapy and undertake a complete baselineclinical and laboratory evaluation before starting the firstpulse. Haemogram, serum electrolytes, renal and liverfunction tests, blood sugar (including HbA1C), urinemicroscopic examination, chest X-ray, electrocardiogram andpregnancy test are some of the preliminary laboratory teststo be done at the first visit of the patient. Blood sugar,serum electrolytes, urine microscopic examination, bodyweight and blood pressure should be monitored at base lineand at each visit of the patient.

Adverse effects of DCP therapyThe adverse effects of pulse therapy are those of its

constituent drugs. Corticosteroids precipitate infections,diabetes mellitus, hypertension, hyperacidity andosteonecrosis. Side effects attributed to cyclophosphamideare leucopenia, hematuria, gonadal failure, hyperpigmentation and hair loss. These side effects are infrequentcompared to daily corticosteroid therapy. Side effectspeculiar to pulse therapy include hiccups 11, facial flushing12, diarrhea, weakness, generalized swelling and weight gain,joint and muscle pains, 13 arrhythmias and shock. Arrhythmiais attributable to rapid efflux of potassium and influx ofsodium in the myocardium.

MINI PULSE CORTICOSTEROID THERAPYOral betamethasone has been given at a dose of 10 mg

once weekly in dermatoses like vitiligo, lichen planus,alopecia areata with variable success. 10 mg ofbetamethasone is split in 2 equal doses on 2 consecutivedays a week.

OTHER FORMS OF PULSE THERAPY

1. Cyclophosphamide pulse therapy

Gokhale et al 14 evaluated the response to pulseintravenous cyclophosphamide therapy in patients ofpemphigus vulgaris. Cyclophosphamide 500 mg pulsesare given in 500 ml of 5% dextrose solution slowlyintravenously. These pulses are given monthly for12 months and 2 monthly for further 6 pulses. Closemonitoring of kidney and bladder function was done. Theresponse to therapy was good to excellent in more than60% of patients. This form of pulse therapy has been triedin other conditions like lupus nephritis, serious centralnervous involvement in lupus erythematosus andWegener’s granulomatosis.

2. Pulse therapy with antifungalsItraconazole, fluconazole are used to treat superficial

and deep fungal infections. These drugs are administered inweekly doses every month.

Itraconazole - 400 mg / day for 1 week every month for3 months, for treatment of Tinea unguium.

Fluconazole - 400 mg once a week for 1 month, fortreatment of Pityriasis versicolor

Each pulse is given for 7 days every month.

3. Isotretinoin therapy for acne vulgaris1 mg / kg / day of isotretinoin is administered for 7-

10days every month.

4. Pulse therapy in PsoriasisWeinstein Frost regimen - Methotrexate 7.5 mg – 25mg

,administered every week. It is used for the treatment ofpsoriasis

CONCLUSION:Pulse therapy appears to be novel path breaking therapy

for pemphigus vulgaris, a host of autoimmune dermatosesand systemic diseases. In India, maximum work has beendone on pulse therapy in pemphigus vulgaris. However, thesteroid molecule used is dexamethasone, not methylpredinolone. Pharmacokinetic and immunologic studies aregrossly lacking in Indian perspectives which would help usto understand the mechanism of action and refine this pathbreaking therapy further. For practical purposes, everyevaluation of pulse therapy should address few clinicaloutcomes: the time to clinical remission, the duration ofremission while on treatment and duration of remission afterwithdrawal of treatment.

REFERENCES:

1) Bell PR, Breggs JD, Calman KC, Patron AM, WoodRF, Macpherson SG et al. Reversal of acute clinical andexperimental organ rejection using large doses of intravenousprednisolone. Lancet 1971; i: 876-80.

Short Review


2) Kountz SL, Cohn R. Initial treatment of renal allograftswith large intrarenal doses of immunosuppressive drugs.Lancet 1969; i : 338-40.

3) Liebling MR Leib E, Mc Laughlin K Blocka K, FurstDE, Nyman K, et al. Pulse methylprednisolone in rheumatoidarthritis. Ann Int Med 1981; 94:21-6.

4) Cathcart ES, Idelson BA, Scheinberg MA, Couser WG.Beneficial effects of methylprednisolone “pulse” therapy indiffuse proliferative lupus nephritis. Lancet 1976; 1:163-66.

5) Pasricha JS, Srivastava G. Cure in Pemphigus apossibility. Indian J Dermatol Venereol Leprol 1986; 52:185-6.

6) Kaur S, Kanwar AJ. Dexamethasone cyclophosphamidepulse therapy in pemphigus. Int. J Dermatol 1990; 29:371-74.

7) Becker LR, Bastian BC, Wesselmann U, Karl S, HammH, Brocker EB. Paraneoplastic pemphigus treated withdexamethasone / cyclophosphamide pulse therapy. Eur JDermatol 1998; 8: 551-3.

8) M Ramam. Dexamethasone pulse therapy indermatology. Ind J Dermatol, Venereol, Leprol 2003 ;69(5):319-22.

9) Pasricha JS. Pulse therapy as a cure for autoimmunediseases. Ind J Dermatol, Venereol, Leprol 2003 ; 69(5):323 - 28

10) Rao P.N, Lakshmi TSS. Pulse therapy and itsmodifications in pemphigus: A six year study. Indian JDermatol Venereol Leprol 2003; 69(5): 329-33.

11) Kanwar AJ, Kaur S, Dhar S, Ghosh S. Hiccup – a sideeffect of pulse therapy. Dermatology 1993; 187: 279.

12) Dhar S, Kanwar AJ, Facial flushing – a side effect ofpulse therapy. Dermatology 1994; 188: 332.

13) Appelhans M, Monsmann G, Orge c, Brocker EB,Dexamethasone – cyclophosphamide pulse therapy inbullous autoimmune dermatoses. Hautarzt 1993; 44:143-47.

14) Gokhale Neeta R et al. Treatment of pemphigus withintravenous pulse cyclophosphamide. Indian J DermatolVenereol Leprol 2003; 69 (5):334-37.

Short Review


TOOTH WITHIN A TOOTHBalagopal Sundaresan a, Arathi Ganesh a, Nivedha Rajendran a

ABSTRACTEndodontic treatment for teeth that exhibit the dental

anomaly called “Dens Invaginatus” is very difficult due tothe operator’s inaccessibility to the diseased pulp in thecomplex root anatomy. Surgical intervention and extractionare the common approaches to deal with this condition.


Dr. BALAGOPAL SUNDARESANVice - Principal and Professor & Head,Department of Conservative Dentistry and Endodontics,Sri Ramachandra Dental College and Hospitals,Porur, Chennai - 600 116email id: [email protected] Dept. of Conservative Dentistry & Endodontics

This case report describes a dens invaginatus with bizarreroot canal anatomy which was successfully treated by non– surgical endodontics.

Key words: Dens in dente, Dens Invaginatus, case reports.

INTRODUCTION‘Dens in dente’ meaning ‘tooth within a tooth’ is a

rare developmental anomaly of the tooth affecting mostlythe permanent maxillary lateral incisors and less commonlythe other permanent teeth and the primary dentition. It isalso called as ‘dens invaginatus’ or ‘dilated compositeodontome’. This condition is characterized by theinfolding or inversion of the enamel into the toothstructure, during the early stages of tooth bud formation.This gives an appearance of an in – growing tooth.[1] Densinvaginatus is commonly diagnosed as an incidentalradiographic finding unless the patient presents with painor swelling associated with the involved tooth. Severeform of dens in dente affects the entire morphologicstructure of the tooth. The incidence of dens invaginatusis reported to range between 0.04% and 10% [2] with ahigher male predilection than females.

Several causes of this condition have been proposed.These include localized external pressure, focal growthretardation and focal growth stimulation in certain areas ofthe tooth bud. Roentgenographically it is recognized as apear – shaped invagination of the enamel and dentin with anarrow constriction at the opening on the surface of thetooth. The significance of dens in dente is its predispositionto early decay, pulp necrosis and periapical cyst. [3] This isbelieved to be due to the patients inability to keep thedefect free of cariogenic plaque. [4]

Oehlers FAC[5] in 1957 classified this defect into threepossible variations:

Type I: The enamel invagination is limited to the dentalcrown short of the cementoenamel junction(CEJ).

Type II: The defect penetrates beyond the CEJ into the dentalroot; the pulp chamber may or may not be involved

Type III:The invagination extends beyond CEJ reaches theperiodontal tissues and has a separate apical foramencommunicating with the periapical tissues

Management of malformation aims to preventdegeneration of the invaginated enamel. Variations such astype II and type III require complicated endodontic orsurgical intervention. This case report describes the non –surgical management of a complicated type III variant ofdens in dente.

CASE REPORTA 22- year – old female presented with swelling and

pain in the gums in the anterior region of the upper jaw(Patient reported on 7th April 2005). The patient gave ahistory of recurrent swelling in the same region for the pastsix months which subsided on its own. On examination amild firm swelling was present in the gingiva in relation tothe upper left lateral incisor. A sinus tract opened in thefacial gingiva in relation to this tooth. There was no evidenceof pus discharge from this sinus tract. The tooth exhibited adifferent external morphology resembling a conical tooth[Fig 1] unlike the trapezoidal morphology of a normal lateralincisor [Fig 2]. It was tender on percussion and showeddelayed response to tooth vitality tests which indicated thatthe tooth was turning non – vital.

Fig 1: Conical appearance of the Dens in Dent lateral incisor

Fig 2: Trapezoidal appearance of a normal lateral incisor

Case Report


The radiographic examination revealed that the tooth wasinvaginated till the apical third of the root with the presenceof periapical radiolucency. The root canal morphologyappeared more complex radiographically for the invaginatedtooth [Fig 3] unlike a normal lateral incisor [Fig 4]. This ledus to diagnose the tooth as type III dens invaginatus withperiapical abcess.

Fig 3: Radiographic appearance of dens in dente

Fig 4: Radiographic appearance of normal lateral incisor

Root canal treatment was initiated for the lateral incisor.On opening the access to the root canal, two canals mesialto and separate from the main root canal system were present[Fig 5]. This finding is in contrast to the normal morphologyof a maxillary lateral incisor which has only one root canal[Fig 6]. All the three root canals in this tooth were separatefrom each other throughout its length. Mild pus dischargewas evident from all the canals suggesting the presence ofan abscess in the periapical tissues.

Fig 5: Access opening of dens in dente showing three rootcanals

Fig 6: Access opening of normal lateral incisor showing onelarge root canal

Root canal treatment was completed in two visits withan inter-appointment dressing of calcium hydroxide.Radiographs were taken post operatively to confirm theintegrity of the root canal filling. Figure 7 shows the postoperative sixth month follow-up radiographic appearanceof the filled root canals of the invaginated tooth showingsatisfactory bone healing periapically. This can be comparedwith the radiographic appearance of a root canal treatednormal lateral incisor with single canal in figure 8. At thesecond appointment two weeks later, the sinus tract wasfound to be completely healed and there was no evidenceof swelling in the gingiva. The final restoration of the toothwas done and then patient was reviewed for clinical andradiological signs of healing after 1, 3 and 6months (October2005). The patient was asymptomatic and did not have anyrecurrence of symptoms in the affected tooth.

Fig 7: Radiographic appearance of obturated (filled) dens indente showing three canals (sixth month follow-up)

Fig 8: Radiographic appearance of obturated (filled) normallateral incisor showing one root canal

Case Report


DISCUSSIONBacterial contamination of the invagination resulted

in development of infection and periapical inflammation.Treatment of dens invaginatus is a complex procedurebecause of its atypical root canal anatomy. [6] The successof the root canal treatment in these teeth depends onidentifying all the additional canals and restoring the same.This case showed the presence of three root canals insteadof one. Identification of the complex root canal anatomyof this case and management of all the canals non-surgically, has improved the prognosis of the tooth.Calcium hydroxide, used as an inter - appointment dressingin the root canals, helped in healing the periapicalinfection and disinfecting the root canals[6] owing to itsanti – microbial and tissue dissolving properties. Thisresulted in early elimination of the root canal infectionthereby avoiding surgical intervention. The success of thisnon – surgical endodontic treatment of the densinvagination was demonstrated by the resolution of thepatient’s signs and symptoms with radiographs showingperiapical bone healing in the subsequent follow – upexaminations.

CONCLUSIONDens invaginatus can be recognized before the eruption

of the tooth from periapical radiographs. So these teethshould be treated prophylactically as soon as possible aftertooth eruption. Early diagnosis and intervention candefinitely prevent pulpal necrosis and the potential loss oftooth. The nonsurgical endodontic management of thecomplex root canal morphology of these teeth is asuccessful alternative to the more invasive surgical

intervention. Operator skill in locating these abnormalcourses of the root canals will eliminate the proceduralerrors which could occur while searching for the canal inits normal position. Knowledge about such unexpectedvariations in root canal anatomy and its immediateconservative treatment is the key to the successfulmanagement of the anomalies of tooth.

REFERENCES:1) Mauro Caldari, Carlo Monaco, Leonardo Ciocca,Roberto Scotti. Single session treatment of a majorcomplication of dens invaginatus: A case report.Quintessence Int, 2006; 37: 337 – 343.

2) Hovland E, Block R. Non-recognition and subsequentendodontic treatment of dens invaginatus. J Endod, 1977;3: 360 – 362.

3) Tsurumachi T, Hay ashi M, Takeichi O. Non-surgicalRCT of dens invaginatus type 2 in maxillary lateral incisor.Int Endodontic J, 2002; 35: 310 – 314.

4) Joseph A Regezi, James J Sciubba, M. Anthony Pogrel.Atlas of Oral and Maxillofacial Pathology, 1st ed,W.B.Saunders, Pennsylvania, 2000.

5) Oehlers FAC. Dens Invaginatus (dilated compositeodontome) I: Variations of the invagination process andassociated anterior crown forms. Oral Surg Oral Med OralPathol, 1957; 10: 1204 – 1218.

6) Hieke Steffen and Christian Splieth. Conventionaltreatment of dens invaginatus in maxillary lateral incisorwith sinus tract: One year follow up. J Endod, 2005; 31:130 – 133.

Case Report


CORTICAL VENOUS THROMBOSIS IN A CASE OF PREECLAMPSIAUsha Vishwanath a, Sonali Sood a, Shalu Gupta a

INTRODUCTIONPreeclampsia a pregnancy specific syndrome clinically

manifesting as elevated blood pressure and proteinuria isone of the major contributors of maternal morbidity andmortality of pregnant mothers in the developingcountries.(1,2)

World wide, 50,000 women die each year frompreeclampsia and it still remains the most significant andintriguing unsolved problems in obstetrics. (3) Cerebrallesions have rarely been demonstrated with preeclampsiabut more commonly associated with eclampsia,rangefrom cerebral edema, hyperemia, ischemia, thrombosis tohemorrhage (ranging from petechiae to gross bleed). Theselesions may be wide spread, focal and seldom fatal.Cortical cerebral venous thrombosis is a rare but dreadedcomplication of preeclampsia and this case is reported forits rarity and dramatic response to therapy.

CASE REPORTA 23 years old primigravida presented to the

Department of OBGYN in October 2006 with the anomalyscan at 23 weeks showing features suggestive of intrauterinegrowth restriction (symmetrical) with all fetal parametersbelow 5th percentile with severe oligohydroamnios. TheDoppler study showed features suggestive of utero placentaldysfunction and the uterine artery had persistant diastolicnotch and the umbilical artery had absent diastolic flow.There were no obvious identifiable congenital anomalies inthe fetus.

On clinical examination, the patient was moderatelybuilt with normal blood pressure 120/80mmHgSystemic examination was within normal limits. Onabdominal examination there was a lag of > 4 wks withgrossly decreased liquor with fetal heart rate of 156/minby Doppler.

ABSTRACTA 23 Year old primigravida presented to the Obgyn out

patient department with severe early onset fetal growthrestriction and oligohydramnios with persistent diastolicnotch in the uterine artery. Patient subsequently went on todevelop full blown early onset severe preeclampsia whichfurther manifested itself in the post partum period asgeneralized clonic tonic convulsions due to cerebral


Dr. USHA VISHWANATHAssociate Professor - Department of OBGYN,Sri Ramachandra Medical College & Research InstitutePorur, Chennai - 600116.E-mail : [email protected] Department of OBGYN

hemorrhage & infarction with cortical venousthrombosis. This is a rare but dreaded complicationof preeclampsia. This late onset eclampsia was thenmanaged conservatively with anticonvulsants andthrombolytic agents.Key words : pre-eclampsia, postpartum period,venous thrombosis, intrauterine growth retardation,oligohydramnios, case reports.

In view of severe early onset IUGR witholigohydroamnios and uterine artery showing persistentdiastolic notch (which is a very sensitive marker forprediction of preeclampsia -78% sensitivity),the patientwas explained regarding the poor fetal prognosis andpossible maternal complications of continuing thepregnancy and counselled for termination. However thepatient decided to continue the pregnancy and was lostin follow up. Subsequently, in the next 3 weeks shedeveloped severe preeclampsia with uncontrolled bloodpressure, for which termination of pregnancy was done.She expelled a dead fetus(weighing 300 gm with noobvious external congenital anomalies in the foetus andplacenta showed no obvious external areas of infarctionor calcification). After delivery and discharge patient didnot go for postnatal check up and presented to theEmergency department of SRMC & RI with an episode ofgeneralized tonic clonic seizures on the 10th post natalday. The seizures were preceeded by a headache(temporoparietal) for 3 days. The patient was immediatelytreated with parenteral anticonvulsants and antiedemameasures. Investigations for preeclampsia were withinnormal limits. The Fundus showed Grade I hypertensivechanges.

MRI brain was done which showed haemorrhagicinfarcts in the left temporoparietal region (fig 1 and fig 2)and in view of patients history MR venogram wasperformed in the same sitting which revealed left transverse

Fig. 1

Flare Coronal image ShowingHaemorrhagic infarct in leftTemporoparietal Region

Fig. 2

T2 Weighted Axialimage showing Infarct

Case Report


and sigmoid sinus thrombosis(fig 3). Therefore patient startedon therapeutic low molecular weight heparin. Meanwhilepatient was evaluated for thrombophilic disorder, all ofwhich showed negative results. Patient showed drasticimprovement symptomatically. After 5 days of overlap,anticoagulation was changed to Warfarin and the patientwas discharged and is presently doing fine. Repeat MRI hasbeen planned after 6 months for evidence of complete cureand to do antiphospholipid workup after anticoagulant isstopped.

DISCUSSIONPreviously the controversial existence of delayed

postpartum eclampsia is now acknowledged by mostexperts. Evidence suggests the increasing incidence of lateonset eclampsia(6). By definition, convulsions with initialpresentation more than 48 hours but less than 4 weeksafter delivery are commonly referred to as late postpartumeclampsia. The presentation of late postpartumpreeclampsia or eclampsia may differ from that occurringduring pregnancy. This contributes to the difficulty in thediagnosis (4).

Lubrasky and Charms reported 44% and 79% patientsrespectively with late onset postpartum eclampsia who had

Fig. 3MR Venogram showing Thrombosis

of Left Sigmoid and Transverse Sinuses

not been identified as having preeclampsia before the onsetof seizures (5, 6). Late postpartum eclampsia is mostcommonly preceeded by a prodrome of visual and cerebralsymptoms (headache/ diplopia) which should not be ignoredin any post natal patient.

Our patient had convulsions on the 10th postnatal day.The response to anticonvulsants with MRI showinghaemorrhagic infarct with venous thrombosis with Fundusshowing grade I hypertensive changes, with exclusion ofmetabolic, thrombophilic and infectious causes, stronglysupport the diagnosis of ECLAMPSIA.(Spontaneous corticalvenous thrombosis which is a rare occurrence in pregnancyis a possible differential diagnosis for this case)

Lesions in MRI cannot predict whether damage ispermanent or likely to be reversible. To reduce the rate ofpostpartum eclampsia, efforts should be directed to educatethe patient and her health care providers regarding promptreporting and evaluation of symptoms of preeclampsia duringthe postpartum period.

REFERENCES:

1. Geary M. the HELLP Syndrome Br J Obstet Gynaecol1997 ; 104 : 887-91.

2. Robert JM, Redmen CW Preeclampsia : More thanpregnancy induced hypertension. Lancet 1993; 341: 1447-51.

3. Duly L maternal mortality associated with hypertensiondisorders of pregnancy in Africa, Asia, Latin America andthe Caribbean Br J Obstet Gynaecol 1992; 99: 547.

4. AM J of Me med ISSN 6735-6757 volt 23>168-170.

5. Lubrasky SL, Barton JR Freidman SA, Saba BM. Late PPeclampsia revisited. Obstet Gynaecol 1994; 83: 502-5.

6. Late postpartum eclampsia: a preventable disease? AMJObstet Gynaecol 2002; 186: 1147.

Case Report


INTRODUCTIONComplications arising from infection of the paranasal

sinuses still occur in paediatric sinusitis, despite the use ofbroad spectrum antibiotics. Inflammatory orbitalcomplications can cause loss of vision. Orbitalcomplications can be divided into several overlappingstages 1: inflammatory edema, orbital cellulitis, orbitalperiosteitis, subperiosteal abscess, orbital abscess andcavernous sinus thrombosis 2.

Functional endoscopic sinus surgery is an establishedstandard for surgical treatment of infections of the paranasalsinuses.

Case report:A 4 year old boy was referred from the Paediatric

department with complaints of swelling in left eye for 2weeks. The swelling was initially in the medial aspect ofthe left eye and then progressively increased. The swellingwas associated with fever and pain at the onset. Patientwas being treated with antibiotics and steroids as prescribedby the Ophthalmologist and pain and fever subsided butthe swelling persisted. There was no visual disturbance.There was no history of trauma or nasal surgery. Examination

ORBITAL COMPLICATION OF PAEDIATRIC SINUSITIS- A CASE REPORTS.B Jothiramalingam*a, Dinesh Kumar a, Prasanna Kumar a, Vivek Sasindran a

ABSTRACTIntraorbital abscess is a serious complication of

sinusitis. The recommended surgical procedure at presentfor drainage of abscess is endoscopic approach. Here we


Prof. S.B. JOTHIRAMALINGAMDept of ENT, Head and Neck SurgerySri Ramachandra Medical College and Research InstitutePorur, Chennai - 600 116.E-mail: [email protected] Dept. of ENT, Head & Neck Surgery

report a case of complicated paediatric sinusitis which wastreated successfully.

Key words: Sinusitis, Subperiosteal abscess, Orbitalcomplications, case reports.

revealed a small swelling 1 X 1 cm just above the leftmedial canthus with associated edema of the upper eyelid.Reddish discoloration of the whole upper eyelid was noted.The swelling was tense and tender on palpation. The lefteyeball was displaced forwards, downwards and outwards(fig .1 ). All the extraocular movements were normal exceptfor restricted upward gaze. Ophthalmologic examinationrevealed normal fundus with no visual impairment.Diagnostic nasal endoscopy showed enlarged middleturbinate on left side with crowded ostiomeatal complex.Mucoid discharge was seen in choana with adenoidhypertrophy. CT scan of Paranasal Sinuses & Orbit withcontrast showed homogenous opacity involving the wholeof the left maxillary sinus and ethmoidal sinus with

Fig. 1 - Pre-operative picture showing displacement of theleft eyeball forwards, downwards and outwards.

Fig. 2 - CT Scan of the paranasal sinus with constrast showinghomogenous opacity involving the whole of the left maxillarysinus and the ethmoidal sinus with extension into theextraconal compartment. Peripheral enhancement of theabscess can be noted

Case Report

extension into the extraconal compartment of left orbitthrough a defect in the lamina papyracea ( fig. 2 ). Peripheralenhancement of the lesion was noted. With the provisionaldiagnosis of left orbital subperiosteal abscess with orbitalcellulitis, the patient was taken up for endoscopic drainageof the abscess. Under general anaesthesia, using a 0 0 degreeHopkin’s telescope uncinectomy and middle meatalantrostomy was performed on the left side. Polypoidal tissuewas removed from the maxillary antrum and the maxillaryostium was made patent. The ethmoidal air cells werecleared till the lamina papyracea could be visualized. Thepapery thin bone of the lamina was removed almostcompletely and 10 ml of frank pus was drained out andswab was sent for culture and sensitivity. Patient wascontinued on broad spectrum intravenous antibiotics. In


the immediate post operative period, the proptosis subsidedand the eye movements were back to normal. Nasal packswere removed the subsequent day and patient was dischargedafter 5 days of antibiotics and steroids. However the pus didnot grow any organism. Patient has been followed up forthree months with no evidence of recurrence ( fig .3 ).

Discussion:Complications of sinusitis can be life threatening and

often require surgical therapy. Complications originate,from progressive spread of inflammation along the bonydehiscences in the lamina papyracea, directly by infectiousbreakdown of the lamina along the vessels2, and in childrenalong the open suture lines. The commonest sinus to beinvolved in paediatric age group is the ethmoid sinus.Compression of small nutrient vessels of the optic nervemight be responsible for loss of vision 3. The infectiousagents are often Haemophilus Influenza (especially in smallchildren) 4, Streptococcus pneumoniae and Staphylococcusaureus. It is difficult to estimate the incidence of orbitalcomplications because many patients are treated effectivelyby paediatricians or general practitioners. In the initial stagei.e. orbital cellulitis, genuine proptosis of the globe occursand this can be difficult to distinguish from the oedema ofthe lids. The proptosis will be exaggerated by a specificcollection of pus which is most often sub or extraperiosteal.Pus readily collects between the sinus and orbit, strippingthe periosteum from the adjacent lamina papyracea. Theposition of the abscess will determine the angle ofdisplacement, usually combining a degree of axial proptosis

with lateral and inferior displacement, as was seen in ourpatient 5. The movements of the globe may be restrictedby the presence of the mass and/ or oedema of the ocularstructures. Visual loss will depend upon the extent andrapidly with which the displacement develops. Colourvision is often impaired first. Once vision is lost it isexceptional for it to return even after surgical and medicaldecompression. Radiological imaging is mandatory toconfirm the diagnosis and for exact planning of the surgery.

Computed Tomographic scans are the imagingmodality of choice for chronic sinusitis and orbital as wellas further complications such as septic cavernous sinusthrombosis 5,6.

An ophthalmologist consultation to document visualacuity should be performed in every case. Endoscopic sinussurgery is a functional and minimally invasive techniqueand is the treatment of choice at present. The advantages ofendoscopic surgery over external approach are minimalsurgical trauma, no drains, no visible scars and the healingcan be followed by the use of an endoscope. Early diagnosisand intervention is mandatory to prevent life threateningcomplications of sinusitis.

Reference:

1. Hawkins DB, Clark, RW. Orbital involvement inacute sinusitis: lessons from 24 childhood patients. ClinicalPaediatrics. 1977;16:464-471.

2. Chandler JR, Langenbrunner DJ, Stevens ER.Pathogenesis of orbital complications in acute sinusitis.Laryngoscope. 1970;80:1414-1428.

3. Anderson RL, Edwards JJ. Bilateral visual loss afterblepharoplasty. Annals of Plastic Surgery. 1980;5:288-292.

4. Rootman J, Robertson W, Lapointe JS. Inflammatorydiseases. In: Rootman J: Diseases of the orbit:A Multidisciplinary Approach. New york, NY:Lippincott;1988:143-157.

5. Clary R, Eavey R, Weber AL, et al. Orbital celluliteswith abscess formation caused by sinusitis. AnnalsOtorhinolaryngology. 1988;97:211-212.

6. Handler LC, Davey IC, Hill JC, et al. Acute Orbit:differentiation of orbital cellulitis from subperiostealabscess by computerized tomography. Neuroradiology.1991; 33:15-18.

Fig. 3 - Post-operative picture showing normal position ofthe eye

Case Report


LAPAROSCOPIC NEPHRECTOMY IN CHILDREN:A CASE REPORT AND REVIEW OF OUR EXPERIENCERamesh Babu a, Vikram a, Shakir a

INTRODUCTIONLaparoscopy has become the mainstay of treatment for

many conditions in adult surgery and urology. However itwas a bit slow to develop in pediatric surgery and pediatricurology. With the availability of expertise and equipments,more centres worldwide have started using the laparoscopicapproach for the management of pediatric urologicalproblems [1].

The advantages of laparoscopy in children includereduced pain, reduced hospital stay and quick recovery whichin turn reduce complications and help them return toschool soon. However pediatric laparoscopy is not withoutits limitations. Limited space available inside the pediatricabdominal cavity can result in difficulty with manipulationof instruments and dissection. Laparoscopic renal surgerywas initially performed via retroperitoneal route. Howeverthe lack of landmarks and limitation in space made trainingin this approach [2,3] difficult. Transperitoneal approachhas now become more popular for laparoscopic renal surgery.In this article we present a challenging case managed in ourunit and discuss our experience of laparoscopic nephrectomyin children, at SRMC.

CASE REPORTA eleven month old baby boy was referred with recurrent

urinary infections, recurrent epidydymo orchitis and failureto thrive. He was the second child of a consanguineousmarriage, diagnosed antenatally to have left multicysticdysplastic kidney. The other gross abnormality noted at thetime of birth was absence of nipples (athelia) and rightundescended testis (Figure 1a and 1b). In view of this akaryotyping was done which was found to be normal.

ABSTRACTLaparoscopic approaches are fast becoming popular

for pediatric urology problems. Here we report a case ofectopic ureter managed laparoscopically. Also ourexperience with laparoscopic nephrectomy in children isreviewed. A 11 month old boy was referred with UTI andrecurrent left epidydymo orchitis. Antenatally he wasdiagnosed to have left multicystic kidney. Ultrasoundpostnatally confirmed dysplastic left kidney with poorfunction on DTPA scan. An MCU revealed reflux in tothe ectopic ureter. Laparoscopic transperitoneal left


Dr. S. RAMESH BABUAssociate Professor in Pediatric Surgery andConsultant in Pediatric UrologyPediatric Urology Unit, Sri Ramachandra Medical Centre,Sri Ramachandra University, Porur, Chennai 600116Email: [email protected] Department of Pediatric Surgery

nephroureterectomy was performed. At the same sittingright orchidopexy was also performed. The child recoveredwell and is devoid of UTIs. Laparoscopy can be a veryuseful tool to remove distal ureter as low as possiblewithout another incision. It reduces hospital stay andhastens patient recovery. All the six patients whounderwent laparoscopic nephrectomy last year haveperformed well. Our results are comparable to thosepublished in the literature.Key words: Urinary tract infection, child, laparoscopy,nephrectomy, case reports.

There were no other dysmorphic features or othersystemic abnormality. Examination of the scrotum revealedabsence of right testis, the left testis was enlarged and tenderdue to epidydymo orchitis. Hematology and Biochemistryexamination were within normal limits. Ultrasoundabdomen revealed a normal right kidney and dysplastic leftkidney with its dilated ureter running ectopically up to thebladder neck. Renal nuclear scan revealed very poor functionin the dysplastic left kidney (Figure 2). Voidingcystourethrogram revealed a bladder diverticulum and refluxinto the dilated ectopic left ureter. The contrast from thedilated ureter was also seen refluxing into the vas deference

Fig. 1a Fig. 1b

Figure 1. a) Eleven month old boy with dysplastic left kidney,ectopic ureter. Clinical examination revealed absent nipples(athelia) b) Same patient having right undescended testis

Figure 2. DTPA renal scan showing poor functionin the left kidney

Case Report


making it more prone for epidydymo orchitis (Figure 3).In view of these findings it was decided to remove thedysplastic left kidney, ectopic left ureter and also perform aright orchidopexy. It would have normally required threedifferent incisions for all the three steps. With the availabilityof pediatric laparoscopy, it was decided to combine all threevia the laparoscopic approach.

Pneumoperitoneum was established via 10mmumbilical port, inserted by open Hassan’s technique. Twofurther 5 mm ports were placed above and below theumbilical port (Figure 4). After mobilising the left colon,the left kidney was dissected and vessels were clipped. Theureter was traced as low as possible and resected at thelevel of the bladder neck. The right testis which was locatedat the level of deep ring was mobilised and brought downwith the assistance of laparoscopy. The resected kidneyspecimen along with the ectopic ureter was removed viathe umbilical port. Histology confirmed dysplasia. Thepatient was advised to continue prophylactic antibiotics.At one year follow up he was free of urinary infections orepidydymo orchitis. The testis brought down laparoscopicallywas felt in the right place in the scrotum.

Table 1Laparoscopic Nephrectomy in Children; A review of casesperformed at the Pediatric Urology Unit, SRMC during theyear 2006

No Age/sex Diagnosis Procedure

1 11 yr; boy PUV, Valve bladder + Lap nephrectomy +Non functioning Ureterocystoplastykidney

2 14 yr; girl PUJ obstruction + Lap nephrectomyNon functioningkidney

3 5 yr; boy PUJ obstruction + Lap nephrectomyNon functioningkidney

4 12 yr; boy PUJ obstruction + Lap nephrectomyNon functioningkidney

5 11 month; Ectopic Ureter + Lap nephrectomy+boy Multicystic kidney+ Lap Orchidopexy

undescended testis

6 10 yr; girl Multicystic DysplasticKidney Lap nephrectomy

DISCUSSION

Children with urinary infection have to be carefullyevaluated as up to 50% of these children can have underlyingrenal abnormality. Vesicoureteric reflux is the commonestcause of childhood urinary infection [1]. Abnormalities likeectopic ureter should be suspected in the presence ofrecurrent epidydymo orchitis in a boy or constant wettingin a girl. Often multicystic dysplastic kidney is associatedwith ectopic ureter.

Our case depicts a text book description of uretericbud developmental anomaly where, the abnormallydeveloped ureteric bud, leads to ectopic insertion of ureterand recurrent epidydymo orchitis [2-4]. Due to abnormalinduction of nephrogenesis, ectopic ureter is often associatedwith dysplastic or non functional kidney on that side.Laparoscopic nephroureterectomy results in cure of thesymptoms in such patients.

In our institute apart from diagnostic laparoscopy,laparoscopic orchidopexy, laparoscopic varicoceleligation, laparoscopic pyeloplasty and laparoscopicnephrectomy are being performed. Open Hassan’stechnique is always used to avoid any possible damageto viscera by blind insertion of the Veres needle.Transperitoneal approach is preferred as the land marksare better within the peritoneum and space constraintseen with retroperitoneal approach is not there.

Although the operative time was slightly higher at thebeginning (3.5 hours), with the learning curve, the current

Figure 3. Voiding cystourethrogram showing reflux into theectopic left ureter and vas. Bladder diverticulum is also noted

Figure 4. Laparoscopic nephrectomy in progress. Umbilical10mm port has been inserted via open Hassan’s technique.Two further ports have been placed.

A retrospective analysis was performed to identify thechildren who underwent laparoscopic nephrectomy in ourunit over the last one year. Table 1 summarizes the differentage groups, indications, and the outcome. The operatingtime for laparoscopic nephrectomy has declined dramaticallywith the learning curve and is at the moment comparableto that of open nephrectomy (2 hours). No complicationswere encountered in any of these patients.

Case Report


operative time for laparoscopic nephrectomy hasapproached that of open nephrectomy (2hours). Postoperative recovery is excellent and the cost is not higheras the hospital stay is reduced significantly compared toopen surgery.

A review of literature showed that our results arecomparable to those reported in the literature [2-6]. Withthe increasing awareness of patients and physicians about‘key hole surgery’ it is essential that we are able to offer thebest available treatment for our patients. The reported caseand the other cases of laparoscopic nephrectomy show thatpediatric laparoscopy can be successfully performed despitethe young age and SRMC has taken a lead in this directionat the right time.

ACKNOWLEDGEMENTSThe author would like to acknowledge the following

faculty and staff who actively helped in the management ofthe case.

Dr. V.K. Sairam, Pediatric Nephrologist

Prof. Betty Chacko, HOD of Pediatrics

Dr. Prakash Agarwal, Pediatric Laparoscopic Surgeon

Dr. Gayathri, PICU consultant,

REFERENCES:

1. Ramesh Babu, Steinbrecher HA: Urinary Tract Infectionand Vesicoureteric reflux. In PAEDIATRIC SURGERY(2nd Edition) by DM Burge, MD Griffiths, HA Steinbrecherand RA Wheeler Arnold, London 2003.

2. El-Ghoneimi A, Farhat W, Bolduc S, Bagli D, McLorieG, Khoury A. Retroperitoneal laparoscopic vs open partialnephroureterectomy in children BJU Int. 2003 Apr; 91(6):532-5

3. Piaggio L, Franc-Guimond J, Figueroa ETE, Barthold JS,Gonzalez R. Comparison of laparoscopic and open partialnephrectomy for duplication anomalies in children. J Urol.2006 Jun: 175 (6): 2269-73

4. Jesh NK, Metzelder ML, Kuebler JF, Ure BM.Laparoscopic transperitoneal nephrectomy is feasible in thefirst year of life and is not affected by kidney size. J Urol.2006 Sep; 176 (3):1177-9

5. Sydorak RM, Shaul DB. Laparoscopic partialnephrectomy in infants and toddlers. J Pediatr Surg. 2005Dec; 40 (12): 1945-7

6. Horowitz M, Shah SM, Ferzli G, Syad PI, Glassberg KI.Laparoscopic partial upper pole nephrectomy in infants andchildren. BJU Int. 2001 Apr; 87 (6): 514-6

Case Report


INTRODUCTION

Acrodermatitis enteropathica was recognized in 1936,by a Swedish dermatologist Thore Brandt1. Zinc absorptionin young patients with this entity is low, about 2-3%compared with 27 – 65% in normal adults. The cause ofthis specific zinc malabsorption is not known. Thedecreased absorption can be overcome by an oral zinc loadbut also improves with age2.

We report here a case of acrodermatitis enteropathicawho showed a dramatic clinical improvement to oral zinctherapy.

CASE REPORT

A 3 year old, male, child, born normally at full termto 2nd degree consanguinous parents presented with ageneralized, pruritic, eczematous eruption. Birth historywas normal. Skin was apparently normal till 3 months ofage.

At the time of presentation, cutaneous examinationrevealed an eczematous eruption on the extremities, frontof the trunk and periorificial areas (Fig 1). The back of thetrunk was relatively spared. There were erythematous, scaly,

ABSTRACTAcrodermatitis enteropathica is a rare, inherited disorder,

transmitted as an autosomal recessive trait. The clinicalsyndrome is characterized by a phenotypic triad of acraldermatitis, alopecia and diarrhoea. The acral and periorificialdistribution of the rash is recognized as a virtually

pathognomonic cutaneous marker for zinc deficiency. It maybe hereditary or acquired in gastrointestinal disorders or inpatients on prolonged total parenteral nutrition. Therapy withzinc gives an excellent clinical response and reduces mortality.

Key words: acrodermatitis enteropathica, zinc, treatment,case reports.

Case Report

ACRODERMATITIS ENTEROPATHICA – A CASE REPORTRashmi Mittal a, Sudha R a, Murugan S a, Adikrishnan a, Shobana S a, Anandan S a


Dr. RASHMI MITTALAssistant ProfessorDepartment of Dermatology & STDSri Ramachandra Medical College & Research InstitutePorur, Chennai - 600116.E-mail : [email protected] Department of Dermatology

Fig 2. Before treatment.Erosions and crusts on face

Fig 1. Before treatment.Crusted lesions on genitalia, thighs

Fig 3. Before treatment.Widespread erosions on soles, gluteal areas

crusted plaques on the central part of the face (Fig 2).The palms and soles showed exfoliation (Fig3). A few whiteplaques were seen on the tongue which were suggestive ofcandidiasis. There was redness and fissuring at the angles

Fig 4. Before treatment.Extensive nail involvement

of the mouth. All the finger and toe nails showed dystrophy,Beau’s lines and were associated with various degrees ofparonychial inflammation(Fig 4). Scalp hair was sparse,thin and light brown in colour.


Milestones were normal. He was fully immunized forhis age. The child had been exclusively breast fed sincebirth. He developed intolerance (diarrhoea) to cow’s milkwhen weaned at 3 months of age and was started on infantformula feeds. The first sibling, a male child had died ofsimilar complaints at 6 months of age.

Examination revealed an afebrile, alert but irritablechild weighing 12kg. He had a height of 82cm, weight of12kg, chest, arm and head circumference of 52cm, 15cmand 49cm respectively. Systemic examination wasunremarkable. He had average intelligence and linguisticskills. A complete haemogram, liver and renal functiontests were normal. Serum albumin was 3.9gm % and serumglobulin 4.2gm % with a reversal of the albumin:globulin ratio. Chest X-ray was normal. Ultrasonographyof abdomen revealed a polycystic left kidney.Histopathological examination revealed mild intracellularoedema in the upper part of epidermis, parakeratosis,dimunition of granular layer and mild psoriasiformhyperplasia(Fig 7). Urine and stool examination wasnormal. Alkaline phosphatase enzyme was 76 IU/L Plasmazinc level was low 52ug/dL (60 – 120ug/dL).

periorificial distribution of the rash is recognized as avirtually pathognomonic cutaneous marker for zincdeficiency. The cutaneous lesions are psoriasiform orannular, erythematous, scaly, crusted plaques. As thedisease progresses these plaques become vesicobullous,pustular and erosive. Other features include stomatitis,apathy, irritability, growth retardation, failure to thriveand delayed wound healing. Delayed puberty andhypogonadism in developing males are some of the longterm effects. Classic hereditary acrodermatitisenteropathica begins within days to weeks after birth ininfants bottle fed with bovine milk or soon after weaningfrom the breast in older infants. Acquired zinc deficiencyusually occurs in patients receiving prolonged totalparenteral nutrition for inflammatory bowel disease andchronic diarrhoea. It may occur in alcoholic livercirrhosis, pancreatitis, cancer chemotherapy.Acrodermatitis enteropathica needs to be differentiatedfrom other dermatological causes of anogenital rash suchas diaper dermatitis, candidiasis, seborrheic dermatitis,psoriasis. Certain other disorders such as essential fattyacid, carboxylase and amino acid deficiencies may havesimilar cutaneous features and need to be excludedespecially if the lesions persist despite zincsupplementation or relapse frequently. Riboflavindeficiency closely mimics this condition but in additionpresents with a seborrheic dermatitis like picture, cornealvascularisation and interstitial keratitis. Histopathologyof acrodermatitis enteropathica is very non specific andnot an absolute criterion to diagnose this condition.

Zinc has been recognised as a constituent of the humanbody. As it forms an integral part of carbonic anhydrase andmany other highly purified enzymes, its importance inprotein and carbohydrate metabolism is understandable. Zincis present in unmilled cereals, beans, cheese, whole wheatbread, animal protein especially lean red meat, pork. Most

Fig 5. One month after treatment

On the basis of clinical and biochemical findings, wearrived at a diagnosis of acrodermatitis enteropathica. Thepatient was treated with oral zinc sulphate supplements2mg/kg/day, topical corticosteroids, warm saline compresses.Oral fluconazole 50mg daily was given for 1 week. Therewas an overall dramatic response to treatment in 7 days.The child became active and playful by the second day .The skin lesions started healing by the 5th day and completelyhealed in one month (Fig 5,6)

Discussion:

Acrodermatitis enteropathica is a rare, inheriteddisorder, transmitted as an autosomal recessive trait. Theclinical syndrome is characterized by a phenotypic triadof acral dermatitis, alopecia and diarrhoea. The acral and

Fig 6. One month after treatment

Fig 7. Histopathology of skin showing parakeratosis,granular layer dimunition, psoriasiform hyperplasia

(H&E, low power).

Case Report


of the diets provide 10 – 15 ug of zinc per day ( 150 –250umol). High content of phytate and fibre in unleavenedbread (chapatti), cereals and vegetables binds the zinc andproduces a highly insoluble Zn – calcium – phytate complexthat prevents absorption. Leavening of bread with a phytate– splitting enzyme of yeast decreases its phytate content by15 – 25% and increases zinc absorption. Fats tend to dilutezinc from the total diet. A preadolescent child should receiveabout 10mg of zinc in the diet per day. The requirement foradolescent male and female are 15mg and 12mg /dayrespectively3.

Zinc sulphate for acrodermatitis enteropathica was firstintroduced in 1973.4 Oral zinc given in a dose of about2mg/kg/day was found to cure all clinical manifestationsrelated to zinc deficiency within 1 or 2 weeks. In mostinstances dietary supplementation with two to three timesthe RDA in doses of 30 to 55mg of elemental zinc daily isadequate to restore normal zinc status. Dosage is based onthe amount of elemental zinc present in the preparation. Asobserved in a study5 treatment with zinc sulphate 1mg/kg/day helped clear the symptoms in 5 days. The serum zinclevels which were as low as 6ug% increased to 75ug% after3 days of zinc therapy. In yet another case report6, zincsulphate was used in a dosage of 5mg/kg/day . There wasrapid improvement of diarrhoea within 24 hours and theskin lesions within 1 to 2 weeks.

This case emphasizes the need for early diagnosis andprompt treatment required to reverse the condition, reducemortality and prevent the long term consequences of zincdeficiency.

REFERENCES:

1. Brandt T. Dermatitis in children with disturbances ofgeneral condition and absorption of food. Acta dermvenereol (Stockh) 1936; 17:513-46.

2. Weismann K, Hoe S, Kauden L et al. Zinc absorptionin patients suffering from acrodermatitis enteropathica andin normal adults assessed by whole – body countingtechniques. Br J Dermatol 1979; 101 : 573 – 9.

3. Ghai OP, Piyush Gupta, Paul VK. Ghai Essentialpaediatrics 6th edn; 2005: 123.

4. Michaelsson G. zinc therapy in acrodermatitisenteropathica. Acta Derm Venereol (Stockh) 1974; 54 :377 – 81.

5. Sivasubramaniam KN, Henkin RI. Behavioural anddermatological changes and low serum zinc concentrationsin two premature infants after Parenteral alimentation. JPaediat 1978 ; 93 : 847 – 51.

6. Sukhjot Kaur, Thami GP, Kanwar AJ. Acrodermatitisenteropathica in a full term breast fed infant . I Journal ofPaediatrics 2002 ; 69(7): 631-33.

Case Report


UNUSUAL PRESENTATION OF RHINOSPORIDIOSIS - A CASE REPORTAnupma Jyoti Kindo a, J. Kalyani a, Sandhya Sundaram b, Senthil Kannan M c, A. Ravi Kumar c


Dr. ANUPMA JYOTI KINDOAssociate Professor Department of MicrobiologySri Ramachandra Medical College and Research InstitutePorur Chennai - 600 116Mobile : 9382740138 Email : [email protected] Dept. of Microbiologyb Dept of Pathologyc Dept. of ENT, Head & Neck Surgery

INTRODUCTIONRhinosporidium is a cosmopolitan disease of man and

domestic animals endemic in India and Srilanka and ishyperendemic in southern districts of Tamil Nadu.

It usually presents as a polypoidal growth in nasal cavitythat involves anterior part of nasal septum and nasalvestibule[1].

The aim of this paper is to report an interesting unusualcase of rhinosporidiosis.

Case report: A 38 year old male Assistant Engineer byprofession hailing from Ennore, Northern part of Chennaicame to the OPD of the ENT Department with complaintsof foreign body sensation in the throat for past 8 years.Patient was able to feel the sensation whenever he hadan episode of upper respiratory tract infection associatedwith unproductive cough. He complained of recurrentepisodes of nasal obstruction. He would develop throatirritation and cough whenever he tried to feel the mass.

There was no complaint of otalgia, no history ofepistaxis and trauma, No medical procedure done in thepast.

Local examination showed a pinkish pedunculated massmeasuring 3x3 cm hanging from the nasopharynx extendingupto the level of the posterior 1/3rd of the tongue along theposterior pharyngeal wall. It was non pulsating or expansile,did not bleed on touch.

Posterior nasal examination showed bilaterally patenteustachian tube, the right choana was filled with a pinkishmaterial, left choana was free.

An endoscopic excision of the nasopharyngel mass wasdone and the tissue was sent to the Microbiology andPathology Laboratory.

Macroscopic and Microscopic findingsThe specimen on gross examination showed a soft

pink mass with white spots on the surface.

ABSTRACTRhinosporodiosis is a chronic infestation by the fungus

Rhinosporidium seeberi, which predominantly affects themucus membranes of the nose and nasopharynx. We report

a case of rhinosporidoisis with presentation as a massextending up to the nasopharynx.

Key words: Rhinosporidiosis, nasopharynx, case reports.

The KOH mount showed numerous sporangiosporeswith occasional sporangium (fig1). The gram smear showedrounded structures which were consistent withrhinosporidiosis.

Fig 1. The KOH mount showing numerous sporangiosporeswith occasional Sporangium (Magnification 400X)

Haematoxylin and eosin staining of tissue sectionsshowed polypoidal fragments lined by stratified squamousepithelium.The subepithelium showed many globularcysts. Each of these cysts represented a thick walledsporangium containing numerous “daughter spores” indifferent stages of development (fig2). The fibroconnectivestroma showed fibroblasts and myofibroblasts and aninflammatory infiltrate consisting of polymorphs andeosinophils. (fig 3). These changes were characteristic ofrhinosporidiosis.

Fig 2. Tissue sections stained with Haematoxylin and eosinstain showing “daughter spores” in different stages ofdevelopment (Magnification 400X)

Case Report


Fig 3. Tissue staining with haematoxylin and eosin showingcharacteristic features of rhinosporidiosis : the polypoidfibroconnective stroma with sporangiospores. Surroundedby dense inflammatory infilterate consisting of polymorphsand eosinophils (Magnification 200X)

Functional endoscopic sinus surgery was performed andthe patient was discharged on the second day postoperatively.

DiscussionOther than India and SriLanka where this disease is

endemic, it has been recognized in many other parts of theworld, like South America, United states, England, Egyptand South Africa [2].

Mode of transmission is through water or dust, fromwhich the endospores penetrate the nasal cavity mucosa,mature into sporangium within the submucosalcompartment and after maturation burst with release ofsporangia into surrounding tissue[2]. Clinically, the lesion

presents as a polypoid, soft mass, sometimespedunculated, of the nose (primary site of infection),the eye and its adnexa, above all conjunctiva, or theurethra. Larynx, trachea, skin and lung are less frequentlyinvolved. Osteolytic bone infiltration is another clinicalpresentation [3]. Histologically the infected tissuereveals granulomatous reaction (mixed cell granuloma),pseudocystic abscesses and fibrosis around the causativeorganism [3].

Surgical excision is the mainstay of treatment. It hasbeen advocated that a wide surgical margin is necessary toreduce risk of recurrence [4].

Rhinosporidiosis is a condition which both cliniciansand laboratory personnel should keep in mind whenmanaging patients from endemic places. Moreover, it is veryinteresting in such cases to follow the clinical course: Aneventual recurrence of the lesion in our patient would meana true relapse excluding the possibility of a reinfection, moreprobable in the endemic areas.

REFERENCE:1. Emmons CD, Binford CH, Utz JP and Kwon-Chung,K. J.: Medical Mycology, 3rd Edition. Lea & Febiger,Philadelphia,1977: 464-470

2. Thomas T, Gopinath N, Betts RH. Rhinosporidiosis ofthe Bronchus. Br.J Surg 1956; 44: 316-19

3. SaqulainG, Jailisi M. Rhinosporidiosis: presenting as amass in the oral cavity. Pak J Otolaryngology 1996;12: 218-20

4. Cho SS, Loh KS. Rhinosporidiosis an unusual cause ofnasal masses gains prominence. Singapore Med J 2004;45 (5): 224-226

Case Report



Prof. M.K. SUDHAKARProfessor, Dept. of General MedicineSri Ramachandra Medical College & Research InstituteChennai - 600116.E-mail : [email protected] Dept. of General Medicine

A CASE OF REACTIVE THROMBOCYTOSISSudhakar M K a, Senthil N a, Mohini Singh a, Hissham a

INTRODUCTIONThrombocytosis is quite often discovered as an

incidental finding during the laboratory evaluation of anunrelated medical problem.However, when it isdetected, it constitiutes an important diagnosticchallenge1. Thrombocytosis can either be due to a reactiveprocess(secondary thrombocytosis) or it can be due to aclonal bone marrow disorder(primary thrombocytosis orclonal thrombocytosis). It is often exceedingly difficult todifferentiate between the reactive and clonal types ofthrombocytosis on the basis of clinical findings orlaboratory test results. Yet there are fundamental differencesbetween them in terms of cause, pathophysiologicalfeatures, and clinical implications2. Here we report a caseof reactive thrombocytosis with an unusually high plateletcount.

CASE REPORT:

A 26 yr old lady presented to us with history of fever,cough, chest pain,breathlessness of one week durationalong with anorexia and weight loss of one month. Patientdid not have any other symptoms. General survey revealedthat she was conscious, oriented and had a temperature of102 degree. Fahrenheit. Respiratory rate was 33/min, BPwas 130/80 mmHg, PR was 116/min she was pale andhad no significant lymphadenopathy. Respiratory systemrevealed diminished breath sound in the right hemithoraxwith severe intercostal space tenderness. Cardivascular,Abdomen and neurological system examination werenormal.

Chest X ray taken revealed a moderate right sidedpleural effusion (Fig 1).

ABSTRACTThrombocytosis is a laboratory abnormality which may

be encountered on routine evaluation of an unrelatedmedical problem.The degree of elevation of platelet countwas once considered to be an indicator whether thethrombocytosis was reactive or due to a primaryhematological disorder.But, current evidence seems to

indicate that the degree of elevation cannot be used as acriteria to differentiate between the two1.We report a caseof reactive thrombocytosis with unusually high plateletcounts, who did not have evidence of a primaryhematological problem and was identified to have two co-existing pathologies which led to the elevated platelet count.Key words : thrombocytosis, empyema, anemia, case reports.

Case Report

Fig. 1 EMPYEMA

USG guided aspiration revealed the fluid to be frankpus and an ICD was inserted subsequently.Investigationsshowed TLC of 18,600( P- 88, L- 10, M- 2), haemoglobin5.4 gms%, platelets 22.9 lakhs/cu mm, ESR >150mm/hr.RFT, eclectrolytes, LFT and urine examination werenormal.USG abdomen – normal study. Pleural fluidshowed WBC-11000,RBC- 6000 and cultures grewStaphylococcus aureus. Manual platelet counting donerevealed it to be 21.6 lakhs/cu mm. Peripheral smearshowed evidence of microcytic, hypochromic anemia alongwith an MCV of 58. Bone marrow aspiration and biopsydone ( in view of very high platelet counts) showed areactive marrow and no evidence of a primaryhematological problem.

A diagnosis of empyema with iron deficiency anemiawas made along with reactive thrombocytosis. She wastreated with IV cefaperazone and sulbactum , ironsupplements, blood transfusions and other supportivemeasures. Serial monitoring of blood indices revealed asteady fall in the platelet count as the infection and anemiaimproved, thereby underlining the cause of the severethrombocytosis as a combination of severe iron deficiencyanemia along with a severe purulent infection in the formof an empyema. Serial blood indices are given in Fig 2 Aand Fig 2 B


DISCUSSION:The most striking feature of this case is the gross

elevation in the platelet count.The degree of elevation ofplatelet count in this patient was such that further work upfor a primary hematological problem was warranted.However, further investigations in that direction did notreveal any evidence of a primary hematological disorder.Thispatient had a severe infection (empyema) and severe irondeficiency anemia which are well established causes ofreactive thrombocytosis. The combination of these twoillnesses may well have caused a dramatic increase in plateletcount3.

The cause of reactive thrombocytosis has been ascribedto cytokine mediated increased synthesis ofthrombopoietin3 , which in turn leads to increased synthesisof thrombocytes.The various causes of reactivethrombocytosis are given in Table 1.1

We report this case to highlight the following currentconcepts in reactive thrombocytosis: Firstly, the degree ofelevation of platelet count is not an indicator whether thethrombocytosis is primary or secondary. Secondly, themanagement of secondary thrombocytosis is purely directedtowards the inciting cause which in our patient was due toiron deficiency anemia and empyema 4.

0

5

10

15

20

25

Plateletcountlakhs

5.4 gm 7.1 gm 9.8 gm 10.1 gmHemoglobin of the patient in gm / dl

Fig 2 B. Hemoglobin of the patient

Case Report

ACKNOWLEDGEMENT:1. Department of Pathology, SRMC, Chennai.2. Department of Cardiothoracic Surgery, SRMC,

Chennai.

REFERENCES:1. Andrew I. Schafer, M.D. N Engl J Med 2004; Currentconcepts in thrombocytosis; 350:1211-1219

2. Kaushansky K. Regulation of megakaryopoiesis. In:Loscalzo J, Schafer AI, eds. Thrombosis and hemorrhage.3rd ed. Philadelphia: Lippincott Williams & Wilkins,2003:120-39

3. Shiguro A, Suzuki Y, Mito M, et al. Elevation of serumthrombopoietin precedes thrombocytosis in acute infections.Br J Haematol 2002;116:612-618.

4. Griesshammer M, Bangerter M, Sauer T, Wennauer R,Bergmann L, Heimpel H. Aetiology and clinical significanceof thrombocytosis: analysis of 732 patients with an elevatedplatelet count. J Intern Med 1999;245:295-300

0

5

10

15

20

25

Plateletcount in

lakhs

18600 14200 11200 9800Total count of the patient - cells

/ mm3

Fig 2 A. Total count of the patient


ERRATUM

The following corrections are made for the article “Prevalence of extended spectrum βββββ-lactam resistance among bacteriacausing hospital infections - detection using PCR”

TYPOGRAPHICAL ERRORS:

1. “bla” genes to read as “blaTEM”and “blasHV”

2. All names of micro organisms to have appeared in italics. The typographical errors in this article are regretted.

3. The pictures with the following legends are published in this issue

4. Page 14, Para 3, Line 16 references to read as “8,9 and 11” and line 20, references to read as “6, 12”.

The above corrections are incorporated in the article and posted in website www.srmc.edu

FIG. 1: Presence of a plasmid (s) seen in drug resistant clinical isolates of E. coli (EC) seen in panel (a)- lanes2,6,10:SRMC 26; lanes4,8- SRMC 3; and in panel (b)- lanes 4,5,6 – R1759,U8688 & E4159. Plasmid(s) of Klebsiella (Kl) areshown in panel (a)- lanes 3,7 - SRMC 20; lane 5, 9 - ATCC 70603 (with plasmid); and in panel (b)- lane 2, 3 & 7 -R1738; R1756 & E4146. The lane 1 in both panels contained ë DNA / Hin dIII digest. Note the presence of more thanone plasmid in some isolates. EC

FIG. 2: A comparison of the sizes of the PCR products on a 1.3% gel from both genes, blaSHV and blaTEM indicating thatthey are of expected lengths. The lanes were loaded with lane 1- 100bp ladder; lane 2- (TEM +ve; 1.2 kb); lane 3- (SHV+ve; 900 bp); lane 4 - negative control. EC- E. coli; Kl- Klebsiella.


FIG. 3: Detection and identification of blaSHV (->) genes by PCR was seen in isolates of E. coli (EC)-lane 8 (panel a) andlane 4 (panel b). Isolates of Klebsieela (Kl) that were positive ca be seen in lane 9 (panel a); lane 3,6,7 (panel b). Lane1- Lambda DNA/Hind III digest.

FIG. 4: Detection and identification of blaTEM (->) genes by PCR seen in panel (a) of isolates of E. coli (EC)- lane 3,4and in isolates of Klebsiella (Kl)- lane 5, 6. A positive E. coli sample (SRMC - 26) showed a~1.2 Kb product (panelb). Lane 1: Lambda DNA/Hind III digest (panel a) or a 1.0 Kb ladder (panel b).


EDITORIAL STATEMENT

Sri Ramachandra Journal of Medicine is the official Journal of Sri Ramachandra

University. Scholarly articles and Case reports which are peer reviewed form the scientific

content of the journal. Views expressed in articles are those of the respective authors and are

not attributable to the Editors.

Website: Sri Ramachandra Journal of Medicine can be accessed online at :

www.srmc.edu./srjm

KIND ATTENTION TO SUBSCRIBERS

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The subscriptions for Sri Ramachandra Journal of Medicine are being serviced at the following

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Please send the subscriptions to the above address. Demand Draft should be drawn in favour of“The Registrar, Sri Ramachandra University, payable at Chennai

jan_2007

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